Compositions and methods for inhibiting G protein signaling

ABSTRACT

Disclosed are compositions and methods for treating diseases associated with G protein βγ subunit activity.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a 35 U.S.C. §371 national phase applicationfrom, and claiming priority to, International Application No.PCT/US2008/061757, filed Apr. 28, 2008, and published under PCT Article21(2) in English, which claims priority to U.S. Provisional PatentApplication No. 60/914,659, filed Apr. 27, 2007, which applications areincorporated by reference herein in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in the course of research sponsored by theNational Institutes of Health Grant Nos. GM60286, DK46371, andK05-DA00360. The U.S. government has certain rights in this invention.

I. BACKGROUND

Five mammalian isoforms of the G protein β subunit (37 kDa) and twelveisoforms of G protein Y (7.8 kDa) have been identified (Offermanns(2003) Prog. Biophys. MoI. Biol 83:101-30). Obligate heterodimerscomposed of G protein β and Y subunits (Gβγ) function as regulatorymolecules in various pathways in eukaryotic cells (Neves, et al. (2002)Science 296:1636-9; Clapham and Neer (1997) Annu. Rev. Pharmacol.Toxicol. 37:167-203). First characterized as a guanine nucleotidedissociation inhibitor (GDI) , Gβγ associates tightly with GDP-bound Gprotein α subunits (Ga) and thereby constitutes the basal form of the Gprotein heterotrimer in which neither Ga nor GβY are active insignaling. Agonist-stimulated G protein coupled receptors (GPCRs)catalyze the exchange of GDP for GTP upon Ga and release of Gβγ from theheterotrimer complex, liberating two active signaling species: Gaα>>GTPand Gβγ. Targets of Gβγ signaling include the G protein-regulatedinward-rectifying potassium channel (GIRK) (Krapivinsky, et al. (1993)J. Biol. Chem. 273:16946-52); type I, type II, and type IV isoforms ofadenylyl cyclase (Tang and Gilman(1991) Science 254:1500-3; Sunahara, etal. (1996) Annu. Rev. Pharmacol. Toxicol. 36:461-80); mitogen-activatedprotein kinase (MAPK) (Schwindinger and Robishaw (2001) Oncogene20:1653-60); phosphotidylinositol-3-kinase (PI3K) (Schwindinger andRobishaw (2001) supra); phosducin (Schulz (2001) Pharmacol Res 43:1-10);at least two members of the G protein receptor kinase (GRK) family(Koch, et al. (1993) J. Biol. Chem. 268:8256-60; Inglese, et al. (1994)Proc. Natl. Acad. Sci. USA 91:3637-41); and other plextrinhomology (PH)domain-containing proteins including the dynamins (Lin, et al. (1998)Proc. Natl. Acad. Sci. USA 95:5057-60; Scaife and Margolis (1997) CellSignal 9:395-401) and the β1, β2, and β3 isoforms of phospholipase C β(PLC β) (Sternweis and Smrcka (1992) Trends Biochem. Sci. 17:502-6; Li,et al. (1998) J. Biol. Chem. 273:16265-72) and many others.

Gβ is a cone-shaped toroidal structure composed of seven four-strandedβ-sheets arranged radially about a central axis (Wall, et al. (1995)Cell 83:1047-58; Lambright, et al. (1996) Nature 379:311-9). Eachβ-sheet is formed from elements of two consecutive WD-40 repeats, namedfor a conserved C-terminal Trp-Asp sequence in each repeat (Gettemans,et al. (2003) Sci STKE 2003:PE27). The GY subunit, an extended helicalmolecule, is nested in a hydrophobic channel that runs across the baseof the cone. The slightly narrower, “top” surface of the Gβ cone is themain binding site of Ga (through its switch II region) (Wall, et al.(1995) supra; Lambright, et al. (1996) supra), phosducin (Loew, et al.(1998) Structure 6:1007-19; Gaudet, et al. (1996) Cell 87:577-88), andGRK2 (Lodowski, et al. (2003) Science 300:1256-62), as shown by thecrystal structures of these complexes. Mutational analysis indicatesthat many interaction partners of Gβy, including PLC β2 and adenylylcyclase, bind to the same surface (Li, et al. (1998) supra; Ford, et al.(1998) Science 280:1271-4). Sites located along the sides of the Gβtorus serve as auxiliary binding surfaces that are specificallyrecognized by certain Gβy targets, exemplified in the crystal structuresof Ga and phosducin bound to Gβy (Wall, et al. (1995) supra; Loew, etal. (1998) supra; Gaudet, et al. (1996) supra; Wall, et al. (1998)Structure 6:1169-83).

Phage display of randomized peptide libraries has been used to identifysequence requirements for binding and screen for peptide that bind toGβ1γ₂ dimers (Scott, et al.(2001) EMBO J. 20:767-76). Although billionsof individual clones were screened, most of the peptides that boundGβχγ₂ could be classified into four, unrelated groups based on aminoacid sequence. One of these groups included a linear peptide (the^(ΛΛ)SIRK″ peptide) with the sequenceSer-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp (SEQ IDN0:1). The SIRK peptide inhibited PLC β2 activation by Gβ1γ₂ subunitswith an IC₅₀ of 5 μM and blocked activation of PI3K. In contrast, theSIRK peptide had little or no effect on Gβ₁γ₂ regulation of type Iadenylyl cyclase or voltage-gated N-type Ca⁺⁺ channel activity (Scott,et al. (2001) supra). This demonstrated that selective inhibition of Gβγbinding partners could be achieved. Peptides belonging to all fourgroups competed with each other with a range of affinities for bindingto Gβ₁γ₂, suggesting that all of the clones isolated from the phagedisplay screen shared a common binding site on Gβ1γ₂ (Scott, et al.(2001) supra).

Subsequent experiments have shown that not only does the SIRK peptideblock heterotrimer formation, but it also displaces Gαn from aGβ1γ₂>>Gαii complex in the absence of Gαii activation and activates Gprotein-dependent ERK1 and ERK2 pathways in intact cells (Ghosh, et al.(2003) J. Biol. Chem. 278:34747-50; Goubaeva, et al. (2003) J. Biol.Chem. 278:19634-41). In vitro experiments revealed that SIRK facilitatednucleotide exchange-independent-A-heterotrimer dissociation (Goubaeva,et al. (2003) supra; Ghosh, et al. (2003) supra) potentially explainingthe activation of ERK in intact cells. Other Gβγ binding peptides suchas QEHA, derived from adenylyl cyclase II (Weng, et al. (1996) J. Biol.Chem. 271:26445-26448; Chen, et al. (1997) Proc. Natl. Acad. Sci. USA94:2711-2714) and amino acids 643-670 from the C-terminal region ofβARK(GRK2) (Koch, et al. (1993) supra) could not promote dissociation ofthe heterotrimer, despite competing for Ga subunit binding (Ghosh, etal. (2003) supra). This indicates that competition for Gα-Gβγ subunitbinding is not sufficient for these peptides to accelerate subunitdissociation.

Using a doping mutagenesis and rescreening strategy, a peptide similarto the SIRK peptide was derived that had higher affinity for Gβ₁γ₂. Thesequence of this peptide isSer-Ile-Gly-Lys-Ala-Phe-Lys-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp (SEQ IDN0:2) (SIGK). In vitro studies with the SIGK peptide indicate that ittoo can displace Gαn from a heterotrimeric complex and also effectivelyprevents heterotrimer formation (Ghosh, et al. (2003) supra). Themechanism by which SIRK/SIGK mediates the dissociation of Gαii* GDP fromGβ1γ₂ is not fully understood (Ghosh, et al. (2003) supra).

II. SUMMARY

The present invention relates to a method for identifying an agent thatmodulates at least one activity of a G protein. This method involvescontacting a G protein β subunit with a test agent and determiningwhether the agent interacts with at least one amino acid residue of theprotein interaction site of the β subunit thereby identifying an agentthat modulates at least one activity of the G protein.

The present invention also relates to a method for identifying an agentthat binds at least one amino acid residue of the protein interactionsite of the β subunit. The method involves the steps of contacting a Gprotein β subunit with a test agent in the presence of a peptide thatbinds at least one amino acid residue of the protein interaction site ofβ subunit, and determining whether the agent inhibits the binding of thepeptide to the at least one amino acid residue of the proteininteraction site of the β subunit thereby identifying an agent thatbinds at least one amino acid residue of the protein interaction site ofthe β subunit.

The present invention further relates to a method for modulating atleast one activity of a G protein. This method involves contacting a Gprotein with an effective amount of an agent that interacts with atleast one amino acid residue of the protein interaction site of the Gprotein β subunit so that at least one activity of the G protein ismodulated.

The present invention is also a method for preventing or treating adisease or condition involving at least one G protein βy subunitactivity. The method involves administering to a patient having or atrisk of having a disease or condition involving at least one G proteinβγ subunit activity an effective amount of an agent that interacts withat least one amino acid residue of the protein interaction site of the Gprotein β subunit so that the at least one activity of the G protein ismodulated thereby preventing or treating the disease or conditioninvolving the at least one G protein βγ subunit activity. Diseases orconditions which involve G protein βy subunit activities include heartfailure, addiction, inflammation, and opioid tolerance.

A kit for identifying an agent that binds at least one amino acidresidue of the protein interaction site of the β subunit is alsoprovided. The kit of the invention contains a SIGK peptide or SIGKpeptide derivative. Or other of the family of phage display derivedpeptides from the original peptide screen described in Scott et al.2001.

Agents identified in accordance with the screening methods of thepresent invention are further provided, wherein said agents have astructure of Formula I, II, or III.

III. BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments and togetherwith the description illustrate the disclosed compositions and methods.

FIG. 1 shows selective binding and modulation of the βγ “hot spot” bypeptides. FIG. 1A shows single alanine substituted mutants of G β₁ werepurified as β₁γ₂ and tested for SIGK and SCAR binding in the phageELISA. FIG. 1B shows the equilibrium measurements of SIGK and SCARpeptide dependent competition for a subunit binding. Peptides,Fα_(i1)GDP, and β₁γ₂ subunits were mixed for 1 h and Fα_(i1) subunitbinding was measured. FIG. 1C shows dissociation rate measurements of αsubunit dissociation from a preformed Fα_(i1)GDPβ₁γ₂ heterotrimer.Peptides were added at time 0.

FIG. 2 shows that small molecules predicted to bind to the Gβ proteininteraction site can interfere with peptide interactions at the proteininteraction site. 1, control (DMSO); 2, NSC30820; 3, NSC12155; 4,NSC13984; 5, NSC117079; 6, NSC610930; 7, NSC293161; 8, NSC23128; 9,NSC402959; 10, NSC109268; 11, NSC125910; 12, SIGK in DMSO. 20 μM of SIGKor 200 μM of each small molecule were used in the assay.

FIG. 3 shows the identification of small molecule ligands that bind tothe Gβγ “hot spot” A) depiction of the “hot spot” binding surface onGβ₁γ₂ that was targeted in the computational screen. FIG. 3B showscompetition ELISA data for three of the compounds identified in thevirtual screen. Compounds were tested for their ability to inhibitbinding of a phage displaying the peptide SIGKAFKILGYPDYD (Davis et al.,2005; Scott et al., 2001). M119 (▪); M109 (▾); M117 (●). Data for thebinding compounds are summarized in Table 1. FIG. 3C shows structures ofrepresentative βγ-binding compounds. FIG. 3D shows the competition ofM119 for interactions between Gα_(i1) and Gβ₁γ₂. F-α_(i1) and M119 weresimultaneously added to Gβ₁γ₂ immobilized on streptavidin beads. Theamount of bead based fluorescence was assessed by flow cytometry asdescribed (Ghosh et al., 2003; Sarvazyan et al., 1998)

FIG. 4 shows the differential effects of M119 and M201 on βγ-dependentregulation of downstream targets. FIG. 4A shows the effects of M119 andM201 (NSC201400) on Gβγ-activation of PLCβ2. Purified PLCβ2 (0.25 ng)was assayed in the presence (▴) or absence (▪) of 100 nM purified Gβ₁γ₂.FIG. 4B shows the effects of M119 and M201 effects on purified PLCβ3(0.5 ng) activity in the presence (▴) or absence (▪) of 100 nM purifiedGβ₁γ₂. FIG. 4C shows the effects of M119 and M201 on activation of PI3Kγby Gβ₁γ₂. Assays contained 10 ng of purified p101/p110 PI3Kγ heterodimerwith or without 100 nM purified Gβ₁γ₂. Left panel: (▴) 100 nM Gβ₁γ₂ or(▪) no βγ. FIG. 4D shows the effects of M119 and M201 on Gβγ-GRK2interactions. M119 or M201 and 25 nM purified GRK2 were addedsimultaneously to 250 nM biotinylated Gβ₁γ₂ (bGβ₁γ₂) immobilized onstreptavidin agarose. GRK2 was detected with antibody to GRK2. Data arerepresentative (mean ±SEM, except D) repeated at least three times each.FIG. 4E shows that M119 (10 μM) but not M201 (1 μM) blocks PLCβ bindingto biotinylated-Gβ₁γ₂ (bGβ₁γ₂). 30 nM bGβ₁γ₂ was incubated with either30 nM PLCβ2 or PLCβ3 in the presence or absence of M119 or M201 for 16 hat 4° C. before precipitation with avidin agarose and detection withPLCβ2,or Gβ antibodies. FIG. 4F shows that M201 enhances binding ofPLCβ3 to bGβ₁γ₂. Same as 4E with PLCβ3.

FIG. 5 shows the effects of M119 and related compounds on Gβγ signalingin DMSO differentiated HL60 cells. FIG. 5A shows that M119 and relatedcompounds block fMLP-dependent Ca²⁺ release in cells loaded with 1 μMFura2-AM. Cells were pretreated with DMSO or 10 μM compounds for 5 minprior to stimulation with 250 nM fMLP. FIG. 5B shows that there was noeffect of M119 (10 μM) on carbachol-dependent Ca²⁺ release from HEK-293cells stably expressing M3 muscarinic receptors. Normalized peak Ca²⁺responses were pooled from three independent experiments each(mean±SEM). Cells were pretreated with DMSO (▪) or compounds (▴) for 5min prior to stimulation. FIG. 5C is the same as A except 10 μM M201 wastested. FIG. 5D shows pooled data for FIG. 5C. FIG. 5E shows the M119and M201 inhibition of GRK2 translocation. Differentiated HL60 cellswere treated with 10 μM compound prior to stimulation with 250 nM fMLP.Translocation of endogenous GRK2 was determined by Western blotting witha GRK2 antibody and quantitative chemiluminescence. Data are mean±SEMfrom 5 experiments, * P<0.05, **P<0.01 ANOVA. FIG. 5F shows M119 andM158C inhibition of GFP-PHAkt translocation. Differentiated HL60 cellsstably over-expressing GFP-PHAkt were treated with 10 μM compound priorto stimulation with 100 nM fMLP. Translocation of GFP-PHAkt to themembrane was determined by Western blotting with an anti-GFP antibodyand quantitative chemiluminescence. Data are mean±SEM from 4experiments. *** P<0.001 ANOVA. FIG. 5G shows the lack of effect ofM119, 158C and M201 on fMLP-induced ERK1 and ERK2 activation.Differentiated HL60 cells were pretreated with 10 μM of compound priorto stimulation with 1 μM fMLP for 5 min. Levels of phosphorylated andtotal ERK were determined by Western blotting.

FIG. 6 shows inhibition of PLC-β2 and PLC-β3 activation in the presenceof exemplary compounds of the instant invention.

FIG. 7 shows compounds related to M119 that inhibit SIGK binding in thephage ELISA. Shown with each compound is the IC₅₀ from the phage ELISA.

FIG. 8 shows small molecule binding profiles. FIG. 8A shows thestructures of M119, M119B, gallein, and fluorescein are shown. FIG. 8Bshows that M119 and gallein bind with comparable affinities in thecompetition phage ELISA. M119 and gallein were tested for their abilityto inhibit binding of a phage displaying the peptide SIGK to the Gβγ“hot spot” as described previously (Bonacci et al., 2006). Data shown isrepresentative of three independent experiments, each in duplicate,±S.D. FIG. 8C shows direct binding analysis of gallein bind to Gβγ bySPR. A representative experiment for gallein binding to bGβ₁γ₂. Galleinbinding was tested at sequentially higher micromolar concentrationsindicated at the peak of each association followed by a dissociationphase with the compound removed between each addition. All data were fitwith a kinetic titration model (Karlsson et al., 2006) to give k_(a) andk_(d) values. In the experiment shown, the fits resulted ink_(a)=1130±17 M⁻¹ s⁻¹ and k_(d)=4.3±0.04×10 ⁻⁴ s⁻¹. Pooled data fromthree separate experiments are given in Table 21.

FIG. 9 shows that “Hot spot” binding small molecules modulate keyleukocyte functions. FIG. 9A shows that M119 and gallein inhibitGFP-PH-Akt translocation. Differentiated HL60 cells stably expressingGFP-PH-Akt were challenged with 250 nM fMLP in the presence and absenceof 10 μM concentrations of the indicated compounds. Translocation ofGFP-PH-Akt to the plasma membrane was evaluated by Western blot.Quantification shown below. ***, P<0.001 analysis of variance (ANOVA) isstatistically different from control (PBS+vehicle). Western blot shownis representative of three independent experiments and quantitationcontains data pooled from three independent experiments±S.E.M. FIG. 9Bshows that M119 and gallein block activation of Rac1. DifferentiatedHL60 cells were challenged with 1 μM fMLP in the presence and absence of10 μM concentrations of the indicated compounds. Rac1 activation wasassessed by Western blots of affinity-precipitated GTP-Rac1 from HL60cell lysates. Western blot shown is representative of three independentexperiments and quantitation contains data pooled from three independentexperiments±S.E.M. *P<0.05 ANOVA is statistically different from control(DMSO only). n=3. FIGS. 9C and 9D shows that M119 and gallein inhibitsuperoxide production in fMLP-challenged HL60 cells. Differentiated HL60cells were challenged with 250 nM fMLP or 250 nM PMA in the presence andabsence of 10 μM compounds or 100 nM Wortmannin (Wtmn). NADPH oxidaseactivity was determined after reaction with NBT by absorbance at 540 nm.Data shown is contains data pooled from three independent experiments(each in duplicate)±S.E.M. *, P<0.05 ANOVA is statistically differentfrom control (DMSO only).

FIG. 10 shows Gβγ “hot spot” binding small molecules inhibit neutrophilrecruitment and acute phase inflammation in vivo. FIG. 10A shows galleininhibition of carrageenan-induced paw edema. Male mice (35-40 g) wereinjected i.p. with 100 mg/kg gallein or 2.5 mg/kg indomethacin in PBS 1h before subplantar injection of 50 μl of 2% carrageenan (Cg) into thetest paw. The contralateral paw was injected with saline as control.Each paw was measured 3 times every 2 h. Change in paw thickness wasquantified by subtracting the average thickness of the contralateral pawfrom the average thickness of the test paw. Each point represents theaverage paw thickness of four mice, each measurement done in duplicate.Data shown is representative of more than three independent experiments.Data are mean±S.E.M. FIG. 10B shows that gallein inhibitscarrageenan-induced paw edema in a dose-dependent manner. Mice (fourmice per plotted point) were treated and quantified as described above.Data are mean±S.E.M. FIG. 10C shows that neutrophil recruitment isattenuated by gallein. Mice (four mice per plotted point) were treatedas described above. Two hours after carrageenan injection, paws weresevered and the number of neutrophils contained within edematous fluidwas determined. Data are mean±S.E.M. ***, P<0.001 and **, P<0.01 ANOVAare statistically different from control. FIG. 10D shows that pawswelling is reduced by gallein. Mice (four mice per plotted point) weretreated as described above. Two hours after carrageenan injection, pawswere severed, and the volume of edematous fluid was determined.***P<0.001 ANOVA is statistically different from control. FIG. 10E showsthe differentiated HL60 cells (200 k) were pre-treated with 10 μM M119and then placed in the upper chamber with 250 nM fMLP in the lowerchamber for one hour. Chemokinesis (CK) was tested using fMLP in boththe upper and lower compartments of the Boyden chamber. n=2, each induplicate. FIG. 10F shows the effects of M119, DL382, and wortmannin onfMLP-induced chemotaxis in primary human neutrophils (expressed as % ofuninhibited fMLP-dependent migration). Same as A with purified humanneutrophils.

FIG. 11 shows that “Hot spot” binding small molecules inhibitGPCR-coupled chemoattract-dependent chemotaxis. FIG. 11A shows that M119and gallein inhibit fMLP-induced chemotaxis in differentiated HL60cells. Differentiated HL60 cells (200 k) were pretreated with 10 μMconcentrations of the indicated compound, challenged with 250 nM fMLP,and assayed for chemotaxis in a Boyden chamber for 1 h at 37° C.Chemotaxis was quantified by counting Diffquik-stained cells in threerandom microscope fields, subtracting out background cells (0-10 cells)in the absence of chemoattractant to obtain total transmigrated cells(˜125 cells fMLP+vehicle), and represented as the percentage offMLP-treated control cells. ***, P<0.001 ANOVA is statisticallydifferent from control. Data shown pooled from three independentexperiments, each in duplicate, ±S.E.M. FIG. 11B shows that neither M119nor gallein blocks 1 μM GM-CSF-induced chemotaxis in a Boyden chamber.Chemotaxis was quantified as above by subtracting out background cells(0-10 cells) in the absence of chemoattractant to obtain totaltransmigrated cells (−100 cells GM-CSF+vehicle) and represented as thepercentage of GM-CSF-treated control cells. No statistically significantdifference from control was seen by ANOVA. Data shown are pooled fromtwo independent experiments, each performed in duplicate, ±S.E.M. FIG.11C shows that M119 and gallein inhibit fMLP- and IL-8-inducedchemotaxis in human neutrophils in a Boyden chamber. Primary humanneutrophils were isolated from whole blood to ≧80% purity. Neutrophils(2×10⁵) were pretreated with gallein (10 μM), M119 (10 μM), M119B (10μM), or wortmannin (wtmn.) (1 μM) and then challenged with 250 nM fMLPor 10 nM IL-8 to evaluate chemotaxis in a Boyden chamber for 1 h at 37°C. Chemotaxis was quantified as above by subtracting out backgroundcells (0-10 cells) to obtain total transmigrated cells (fMLP ˜100 cellsand IL-8 ˜175 cells) and represented as the percentage ofchemoattractant-treated control cells. ***, P<0.001 ANOVA isstatistically different from control. NS, not statistically differentfrom control. Data are mean±S.E.M. Data shown are pooled from threeindependent experiments, each in duplicate, ±S.E.M. FIG. 11D shows thatgallein dose-dependently inhibits human neutrophil chemotaxis in aBoyden chamber. Primary human neutrophils were isolated and treated (250nM fMLP±gallein) as described above. Chemotaxis was quantified as aboveby subtracting out background cells to obtain total transmigrated cellsand represented as the percentage of fMLP-treated control cells. Datashown are pooled from two independent experiments, each in duplicate,±S.E.M.

FIG. 12 shows that Gallein is effective with oral administration. Malemice (35-40 g) were dosed by oral gavage with 30 mg/kg gallein 1 hbefore challenge with 2% carrageenan. Methods are as described in FIG.4. Each bar represents the average paw thickness of four mice at 3 hafter carrageenan injection, each measurement done in duplicate. Dataare mean±S.E.M. ***, P<0.001 and **, P<0.01 ANOVA are statisticallydifferent from vehicle. NS, not statistically different from control.Data shown are representative of two independent experiments.

FIG. 13 shows the antinociceptive effect of morphine (A and B).Wild-type (●, +/+) or mutant (∘, −/−) mice were administered differentdoses of morphine i.p. (A) or i.c.v. (B). Receptor selectivity ofantinociceptive effect (C and D). Morphine was administered i.c.v. towild-type (C) or transgenic (D) mice with or without pretreatment withi.c.v. β-FNA (20 nmol, −24 hr) or co-administered either the x-selectiveantagonist, nor-BNI (3 nmol), or the δ-selective antagonist, ICI 174,864(4 nmol). Antinociception was measured in the mouse tail flick assay 20min after the administration of morphine. Data are presented as the meanpercentage antinociception±SEM. Six to 10 mice were used to obtain eachpoint. *, Significantly different from morphine, P<0.01. Data are fromXie et al. (1999).

FIG. 14 shows shows the effects of M119 on morphine-inducedanti-nociception in A) WT and B) PLCβ3−/− mice. Morphine wasadministered i.c.v. to mice (▪) with or without (▴) 100 nmol M119.Anti-nociception was measured 20 min. after injection using the 55° C.tail flick test. Mean±SEM, 7-10 animals at each point.

FIG. 15 shows that the μ agonists, DAMGO (10 μM) and morphine (10 μM)both significantly increased the total inositol phosphates measured inhMOR-CHO cells, ** p<0.01 compared to control (A). Both M119 (10 μM) andβ-FNA (10 μM) attenuated this response, *p<0.05 compared to the agonistalone. The κ-selective agonist, U50,488 (10 μM) had no significanteffect on IP generation with or without M119 (10 μM) or nor-BNI (10 μM),in the hKOR-CHO cells (B). In the hDOR-CHO cells neither δ-selectiveagonist, DPDPE (10 μM) (C) or Deltorphin II (10 μM) (D) significantlyincreased IP generation over control treated cells. The δ-selectiveantagonist, naltrindole (100 μM), had no effect.

FIG. 16 shows GRK2 expression and activity in non-failing (NF, n=6) andpaired LV samples (n=12) obtained upon LVAD implantation (HF) andsubsequent cardiac transplantation (LVAD). (A) GRK2 proteinimmunoblotting, mean±SEM. Samples from non-failing hearts (NF) were alsoanalyzed (n=6). (+) control is purified GRK2. (B) Soluble cardiaclysates were incubated with [32P-ATP] and purified rod outer segmentmembranes enriched with the GPCR rhodopsin (Rho). incorporation into Rhoafter gel electrophoresis. *, p<0.05 HF vs NF; #, p<0.0005 LVAD vs HF.

FIG. 17 shows that Gβγ blockade with M119 normalizes cardiac functionand reduces pathologic cardiac hypertrophy. FIG. 17A shows M-modeechocardiographic images of conscious adult C57/B16 male mice following1 week of either vehicle or Iso (30 mg/kg/day) delivered by miniosmoticpumps (Veh, . . . or Iso, . . . , respectively) and concurrent dailyvehicle or M119 (100 mg/kg) injections ( . . . , Veh, or . . . , Cmpd,respectively). FIG. 17B shows that animals were treated the same as in(A). Conscious echocardiography was performed on animals prior tosacrifice. % FS is a measure of cardiac contractility derived fromend-diastolic and end-systolic diameters. FIG. 17C shows grossmorphological assessment of HW:BW in animals from (A) and (B). Heartweight to body weight ratio (HW:BW) was assessed upon sacrifice of theanimal. (n=7-8 per condition)

FIG. 18 shows that Gβγ blockade with M119 normalizes GRK2 expression inHF. Immunoblot for GRK2 of equal amounts of total protein isolated fromhearts of adult C57/B16 male mice following 1 week of either vehicle orIso (30 mg/kg/day) delivered by miniosmotic pumps (Veh, . . . or Iso, .. . , respectively) and concurrent daily vehicle or M119 (100 mg/kg)daily injections ( . . . , Veh, or . . . , Cmpd, respectively).

FIG. 19 shows that M119 enhances cardiomyocyte contractility in vitro.FIG. 19A shows representative tracings of untreated isolated adultcardiomyocytes and cells treated with M119, Iso, or M119 and Iso. FIG.19B shows the averaging of 4-7 independent experiments (1experiment=average of≧7 cardiomyocytes per condition) showing that M119treatment significantly increased percent contractility over baselineand enhanced Iso-stimulated contractility. Pretreatment with the generalβ-AR antagonist, propranolol, abolishes the effects of M119 and Iso oncardiomyocyte contractility. FIG. 19C shows that DL382 also increasesthe rate of shortening both in the presence and absence of Iso. *P<.05,**P<.01, ***P<.001

IV. DETAILED DESCRIPTION

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that theyare not limited to specific synthetic methods or specific recombinantbiotechnology methods unless otherwise specified, or to particularreagents unless otherwise specified, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting.

A. Definitions

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that thethroughout the application, data is provided in a number of differentformats, and that this data, represents endpoints and starting points,and ranges for any combination of the data points. For example, if aparticular data point “10” and a particular data point 15 are disclosed,it is understood that greater than, greater than or equal to, less than,less than or equal to, and equal to 10 and 15 are considered disclosedas well as between 10 and 15. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

“Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity,response, condition, disease, or other biological parameter. This caninclude but is not limited to the complete ablation of the activity,response, condition, or disease. This may also include, for example, a10% reduction in the activity, response, condition, or disease ascompared to the native or control level. Thus, the reduction can be a10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction inbetween as compared to native or control levels.

“Treatment,” “treat,” or “treating” mean a method of reducing theeffects of a disease or condition. Treatment can also refer to a methodof reducing the disease or condition itself rather than just thesymptoms. The treatment can be any reduction from native levels and canbe but is not limited to the complete ablation of the disease,condition, or the symptoms of the disease or condition. Therefore, inthe disclosed methods, treatment” can refer to a 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of anestablished disease or the disease progression. For example, a disclosedmethod for reducing the effects of inflammation, treating seizures,treating heart malfunction, or any other method disclosed herein isconsidered to be a treatment if there is a 10% reduction in one or moresymptoms of the disease in a subject with the disease when compared tonative levels in the same subject or control subjects. Thus, thereduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or anyamount of reduction in between as compared to native or control levels.It is understood and herein contemplated that “treatment” does notnecessarily refer to a cure of the disease or condition, but animprovement in the outlook of a disease or condition.

A “decrease” can refer to any change that results in a smaller amount ofa composition or compound, such as AR. Thus, a “decrease” can refer to areduction in an activity. A substance is also understood to decrease thegenetic output of a gene when the genetic output of the gene productwith the substance is less relative to the output of the gene productwithout the substance. Also for example, a decrease can be a change inthe symptoms of a disorder such that the symptoms are less thanpreviously observed.

An “increase” can refer to any change that results in a larger amount ofa composition or compound, such as AR relative to a control. Thus, forexample, an increase in the amount in AR can include but is not limitedto a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% increase.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon.

B. Compositions

Disclosed are the components to be used to prepare the disclosedcompositions as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds may not be explicitlydisclosed, each is specifically contemplated and described herein. Thus,if a class of molecules A, B, and C are disclosed as well as a class ofmolecules D, E, and F and an example of a combination molecule, A-D isdisclosed, then even if each is not individually recited each isindividually and collectively contemplated meaning combinations, A-E,A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed.Likewise, any subset or combination of these is also disclosed. Thus,for example, the sub-group of A-E, B-F, and C-E would be considereddisclosed. This concept applies to all aspects of this applicationincluding, but not limited to, steps in methods of making and using thedisclosed compositions. Thus, if there are a variety of additional stepsthat can be performed it is understood that each of these additionalsteps can be performed with any specific embodiment or combination ofembodiments of the disclosed methods.

The protein interaction site for G proteins has now been appreciated.The structure of Gβγ bound to SIGK was elucidated and indicates thatSIGK binds to Gβγ as an α helix across the Ga interaction surface, in aposition occupied by an α helical region of the switch II domain of Gain the heterotrimer. The conformations of Gβγ in the presence andabsence of SIGK are very similar. Thus, the crystal structure revealshow the peptide blocks Gα-Gβγ interactions. The structure furtherindicates that Gβ has evolved a highly reactive and specialized surfacefor interaction with diverse protein partners. This specialized surfaceis referred to herein as the “protein interaction site” or “proteininteraction site of Gβ.” Analysis of various characteristics of theprotein interaction site led to the understanding that the basis forthis surface as a preferred interaction surface is not an inherentconformational flexibility or unusually high surface accessibility ofthe site, but rather the prevalence of multiple types of potentialinteraction chemistries in this single binding surface. The specificamino acid combinations at this surface required for amino acid sequencerecognition at the protein interaction site have also been determined.Moreover, the specific molecular interactions necessary for eitheracceleration of heterotrimer dissociation or inhibition of proteincomplex formation have been demonstrated.

Accordingly, the present invention relates to a method for identifyingan agent that modulates (i.e., blocks or inhibits, or activates orpotentiates) at least one activity of a G protein by contacting a Gprotein β subunit with a test agent (e.g., in a high-throughput screen)and determining whether the test agent interacts with at least one aminoacid residue of the protein interaction site of the G protein β subunit.A G protein β subunit is intended to include any one of the five knownmammalian G protein β subunit isoforms (Offermanns (2003) supra). Anactivity of a G protein is intended to mean the transduction of signalsthrough the G protein to one or more downstream proteins including, butnot limited to, G protein-regulated inward-rectifying potassium channel(GIRK); type I, type II, and type IV isoforms of adenylyl cyclase;mitogen-activated protein kinase (MAPK) ; phosphotidylinositol-3-kinase(PI3K) ; G protein receptor kinase (GRK) family members; and otherplextrinhomology (PH) domain-containing proteins including the dynaminsand the β1, β2, and β3 isoforms of phospholipase C β (PLC β). Modulationof G protein activity occurs via binding of the agent to at least oneamino acid residue of the protein interaction site thereby blockinginteractions between the Gβγ subunits and Ga subunit or the Gβγ subunitsand the downstream proteins described herein.

The crystal structure of Gβγi bound to SIGK revealed that the SIGKpeptide interacts with residues of Gβ1 subunit that are utilized byseveral Gβy binding proteins {e.g., downstream proteins). For example,Lys57, Tyr59, Trp99, Met101, Leu117, Tyr145, Met188, Asp246, and Trp332of Gβ1 are involved in contacts with the GRK2 PH domain in the crystalstructure of the Gβ1γ₂>>GRK2 complex, and all of these residues of Gβ1are involved in SIGK contacts as well (Table 1). This is in spite of thefact that the secondary-structures of the PH domain that contact Gβ1(the RH-PH loop, the αCT region, and β4 strand) are completelydissimilar to the purely helical SIGK peptide (Lodowski, et al. (2003)supra). This theme is recapitulated in the complex of Gβ1 with phosducin(Ford, et al. (1998) supra) where a common subset of Gβ1 residuescontacts a binding partner with different secondary structure from GRK2.Notably, the switch II region of Gαii forms an α-helix that is bound inalmost the same orientation as the SIGK peptide. However, switch II ofGαn has no sequence similarity to the SIGK peptide, although it containsa lysine (Lys210) which is oriented in almost the same position as Lys4of SIGK (Goubaeva, et al. (2003) supra).

TABLE 1 Gα_(i1) Phosducin GRK2 SIGK PLCβ AC GIRK Ca⁺⁺  42  44  46  47 52  53  55  55  55 55  55  57  57  57 Lys57  57  57  59  59  59 Tyr5959  75  75  75  76  78  78  78 78  78  80 80 80  88  89 89  89 89  90 91  92  95  96  98  98  99  99  99 Trp99 99  99 99 Val100 101 101 101Met101 101 101 101 117 117 117 Leu117 117 117 117 119 119 119 119 132143 143 143 144 145 145 145 Tyr145 162 182 186 186 Asp186 186 186 186188 188 188 Met188 204 204 204 228 223 Asp228 228 228 228  228 230 230Asn230 246 246 246 Asp246 246 246 274 290 290 292 304 310 311 314 314332 332 332 Trp332 332 332 332 41% 44% 44% — 54% 67% 43% 60% Key tocolumn headings: Gαn, the crystal structure of the Gαii^(>>)Gβ1γ₂heterotrimer (Wall, et al. (1995) supra; Wall, et al. (1998) supra);phosducin, the phosducin>>Gβ1γ₂ complex (Gaudet, et al. (1996) supra);GRK2, the GRK2^(>>)Gβ1γ₂ complex (Lodowski, et al. (2003) supra); SIGK,the SIGK^(>>)Gβ1γ₂ complex; PLC β, mutational analysis of the PLCβ2/3^(#)Gβ1Y2 complexes (Li, et al. (1998) supra; Ford, et al. (1998)supra); AC, mutational analysis of the adenylyl cyclase typeI/II^(>>)Gβ1γ₂ complex (Ford, et al. (1998) supra); GIRK, mutationalanalysis of Gβχγ₂ interaction with the GIRK1/4 channels (Ford, et al;i998) supra); Ca⁺⁺, mutational analysis of Gβ1γ₂ interaction with N orP/Q type calcium channels (Ford, et al. (1998) supra; Agler, et al.(2003) J. Gen. Physiol. 121: 495-510). Underlined residues indicateresidues important for the SIGK>>Gβ1γ₂ interaction. The last rowindicates the percentage of residues that are shared between the targetand the SIGK interfaces.

When mutational data for Gβy targets PLC β2, adenylyl cyclase, and GIRKand CCα1B calcium channels are included in this analysis, the footprintof SIGK upon Gβ is similar to the footprints of these former targets(Li, et al. (1998) supra; Ford, et al. (1998) supra). Of the thirteenresidues from Gβ that encompass the protein interaction site, nine(Lys57, Tyr59, Trp99, Met101, Leu117, Tyr145, Met188, Asp246, andTrp332) are also found as contacting residues in the Ga, GRK2, andphosducin complexes (Table 1). These residues reflect a consensus set ofresidues utilized by many Gβ binding partners. An additional three ofthe thirteen residues (Asp186, Asp228, and Asn230) are shared amongstSIGK and two of the other protein complex structures. One of thethirteen, Va1100, contacts SIGK through its main chain oxygen and is notinvolved in binding interactions in the other complexes. The SIGKbinding residues that are most sensitive to mutational perturbation arealso the most frequently involved in interactions with other Gβ bindingpartners. SIGK was identified from a random peptide phage display wheremultiple peptides, unrelated by sequence, appeared to bind to a commonprotein interaction site on Gβa.

Because of the extensive overlap between the residues of Gβ1 that areaccessed by SIGK and those involved in the binding of protein Gβγtargets, SIGK is a competitive inhibitor of multiple Gβγ bindingreactions. The closely related SIRK peptide has effects on severalGβγ-dependent pathways; it blocks Gβγ-mediated activation of PLC β2, PLCβ3 and PI3K in enzyme assays, and induces ERK I/II activation in acell-based assay (Scott, et al. (2001) supra; Goubaeva, et al. (2003)supra). These effects are sensitive to mutations of residues in SIGKthat interact with the surface of Gβ, as Lys4, Ala5, Phe6, Ile8, Leu9,and Gly10 of SIGK have all been shown by alanine scanning to beimportant for inhibition of PLC β2 activation by Gβ1γ₂ (Scott, et al.(2001) supra). In addition, Leu9 of SIGK is important for the ability ofSIGK to activate MAPK pathways in cell culture (Goubaeva, et al. (2003)supra). However, SIRK does not block inhibition of adenylyl cyclase typeI or N-type Ca²⁺ channel regulation, even though their footprints arequite similar to those of Ga and PLC β2 (Scott, et al. (2001) supra).Conversely, mutations in Gβ that abrogate SIGK binding do not equallyaffect interaction with other Gβγ binding partners. For example,mutation of Leu117 to alanine decreases the ability of Gβ1γ₂ to activateadenylyl cyclase type II and PLC β3 and to bind GRK2 and SIGK, but hasno effect on GIRK1/GIRK4 potassium channel activation, CCα1B calciumchannel activation, or PLC β2 activation (Table 1) (Li, et al. (1998)supra; Ford, et al. (1998) supra). Similarly, mutation of Trp332 ofGβ1γ₂ to alanine reduces affinity of Gβ1γ₂ for SIGK and impairsstimulatory activity towards adenylyl cyclase type II, CCα1B and bothPLC β2 and PLC β3, but does not affect interaction with GRK2, activationof GIRK1/GIRK4, or inhibition of adenylyl cyclase type I (Li, et al.(1998) supra; Ford, et al. (1998) supra). Both Leu117 and Trp332 ofGβχγ₂ form part of the Gα_(t) and Gαn binding sites of Gβ1 (Wall, et al.(1995) supra; Lambright, et al. (1996) supra; Wall, et al. (1998) supra)and mutation of Leu117 also affects Gai₁ association with Gβ1γ₂ (Li, etal. (1998) supra; Ford, et al. (1998) supra). Unlike other peptides thatblock heterotrimer formation (Ghosh, et al. (2003) supra), SIGK promotesnucleotide exchange-independent dissociation of Gβ1γ₂ from Gαii (Ghosh,et al. (2003) supra; Goubaeva, et al. (2003) supra). For example, apeptide derived from the C-terminus of GRK2 blocks heterotrimerformation (Ghosh, et al. (2003) supra) but does not promote Gαii*Gβ1γ₂subunit dissociation, even though the structure of the GRK2<<Gβ1γ₂complex indicates that this peptide should utilize much the same surfaceof Gβ1 as SIGK (Lodowski, et al. (2003) supra). Not to be bound bytheory, SIGK could promote heterotrimer dissociation by either of twomechanisms. SIGK may induce conformational changes on Gβ1 that propagatebeyond the SIGK binding site and disrupt other interactions between Gβ1and Gαii. However, the Gβ1γ₂>>SIGK structure shows that SIGK does notinduce substantial conformational change in Gβ1 outside of the SIGKbinding site itself. The second mechanism relies on the assumption thatGαn can dynamically detach from and rebind to either of two surfaces onGβ: the switch II interaction site on the top face of Gβ1, where SIGKbinds in a similar orientation, and the N-terminal interaction surfaceon blade one of Gβ1. Transient release from Gαn at the switch IIinterface would allow SIGK access to Gβ1. Complete release of Gαn fromGβ could then occur if the off-rate for SIGK is slower than that fordissociation of the N-terminus of Gαn. Thus the GRK2 peptide, whichbinds the top surface of Gβ, may dissociate too quickly to promotedissociation of Ga. This dynamic model of Gβγ interactions isbiologically relevant, since many Gβy binding targets exhibit bindingoutside of the top surface of Gβ and may also transiently samplealternate surfaces on Gβ.

The ability of the protein interaction site of Gβ1γ₂ to recognize arange of protein ligands with diverse secondary structures indicatesthat it may be an example of a preferential protein binding site (see,e.g., Delano, et al. (2000) Science 287:1279-1283). Preferential bindingsurfaces are characterized as having high solvent accessibility, lowpolarity, and a large degree of conformational flexibility (Scott, etal. (2001) supra; Ma, et al. (2001) Curr. Opin. Struct. Biol. 11:364-9;Bogan and Thorn (1998) J. MoI. Biol. 280:1-9; Clackson and Wells (1995)Science 267:383-6; DeLano (2002) Curr. Opin. Struct. Biol. 12:14-20).Moreover, preferential binding sites are likely to contain an unusuallyhigh concentration of so-called “hot spots”, i.e., residues that, ifmutated to alanine, reduce binding energy at least ten-fold (DeLano(2002) supra). Hot spots have been described for both protein-proteinand protein-small molecule interfaces; often point mutations to any hotspot on a surface completely abrogate complex formation, even when thebinding interfaces bury several hundred A2 of total surface area (Boganand Thorn (1998) supra; Clackson and Wells (1995) supra; Thanos, et al.(2003) J. Am. Chem. Soc. 125:15280-1; Zhang, et al. (2003) J. Biol.Chem. 278:33097-104). These criteria have been used herein to evaluatethe protein interaction site of Gβ1 as a protein surface that ispredisposed by its chemical composition and surface properties to serveas a protein binding site. Of the twelve residues in the proteininteraction site of Gβ, eight (Lys57, Tyr59, Leu117, Tyr145, Asp186,Met188, Asn230, and Trp332) met the energetic criterion for a hot spotresidue. Replacement of any of these residues by alanine resulted in a10-fold reduction in the affinity of Gβ1Y2 for SIGK. It is clear thatall of these residues act as energetically important nodes thatcontribute favorably to SIGK binding. The SIGK binding surface of Gβ1contains several residues that have been shown to be enriched in hotspots (Bogan and Thorn (1998) supra). These include tyrosine, tryptophanand arginine; bulky residues that are capable of forming both polar andnon-polar interactions. The protein interaction site of Gβ issignificantly more populated with aromatic residues than the rest of theGβ surface. 38% of the SIGK binding surface versus 8.5% of the totalnon-glycine surface accessible Gβ residues is composed of Phe, Tyr, His,or Trp. Therefore, the protein interaction site of Gβ is more nonpolar;in total, 62% of the protein interaction site of Gβ is nonpolar comparedto 29% of Gβ surface accessible residues. Further, asparagine andaspartic acid, which have a moderately favorable distribution among hotspot surfaces, account for four of the thirteen residues in the proteininteraction site of Gβ. This combination of aromatic and chargedresidues allows for accommodation of binding partners with diversechemical properties at the Gβ protein interaction site. Preferentialbinding surfaces are expected to have high surface accessibility(DeLano, et al. (2000) supra). To analyze this property of the proteininteraction site of Gβ, the total surface accessible area was calculatedfor the Gβ molecule on a residue, main chain, and side chain basis. Mostamino acids in the protein interaction site of Gβ were not significantlymore accessible than others of their type in Gβ. However, five residuesshowed significant deviation from the mean: Tyr59, Trp99, Met101,Leu117, and Trp332. In the case of Trp99, side chain surfaceaccessibility was significantly greater than the type average; the mainchain of Tyr59, Trp99, and Met101 were more accessible than the mean.Leu117 had significantly higher main chain and side chain accessibilitythan the mean.

Conformational flexibility or adaptability has been cited as animportant determinant of a preferential binding surface, since suchsurfaces are better able to bind to structurally unrelated proteintargets (DeLano, et al. (2000) supra). Residue flexibility can bequantified in terms of relative positional variation in the context ofseveral protein complexes. Histogram analysis of the RMSD relative touncomplexed Gβ1Yi of all Gβ residues in four crystal structures (Gp₁Y₂′SIGK; Gβ1γ₂#Gαii; Gβ1γ₂>>GRK2; Gβ1Yi#phosducin) shows that the proteininteraction site residues of Gβ exhibit only slightly greater thanaverage side chain positional dispersity (1.42 A compared to 1.35 A),with the side chains of Trp99, Asp228, and Trp332 having the largestpositive deviation from the average (each greater than 2 A). Inparticular, Arg314 and Trp332 in blade seven move more than 10 A towardsthe outside of the Gβ1 torus to interact with phosducin. Atomic Bfactors also provide a measure of conformational flexibility. In thestructure of uncomplexed Gβ1Yi the B factors for Trp99, Va1100, andMet101 exceed the mean value by least one standard deviation (Trp99 isgreater than two standard deviations from the mean). In complexes withGαn, GRK2, phosducin, and SIGK complexes, these binding site residuesbecome more well-ordered with B values close to the mean and in somecases up to one standard deviation below the mean. Thus, the capacity ofGβ to recognize structurally diverse binding partners does not require ahigh degree of conformational flexibility for most residues in theprotein interaction site of Gβ. Small structural adaptations in Gβ1 aresufficient to accommodate a range of co-evolving binding partners.Structural and mutagenic analysis demonstrates that the proteininteraction site on Gβ can be regarded as a hot surface, co-evolved topromote tight binding with multiple protein targets. However, themechanism by which Gβy acts as a hot surface is complex. Trp332 is theonly residue which meets all four of the criteria for a hot spot,although Tyr59 and Trp99 have three of the four characteristics of hotspot residues that were tested. There are other residues in the top faceof Gβ that are sensitive to mutational perturbation and are utilized inmany binding partner interactions but do not exhibit the characteristicsof conformational flexibility, solvent accessibility, or nonpolarityexpected of hot spots. Especially notable among this group are Lys57 andMet188; both of these residues are energetically significant bindingdeterminants in Gβ as shown by mutational analysis and comparison toknown Gβy complex structures, and yet do not meet any of the additionalstatistical criteria for hot spot residues.

Accordingly, an amino acid residue of the protein interaction site of aGβ is intended to include Lys57, Tyr59, Trp99, Va1100, Met101, Leu117,Tyr145, Asp186, Met188, Asp228, Asn230, Asp246, and Trp332. By way ofillustration, the location of these residues is provided in the rat Gβamino acid sequence of:

MGEMEQLKQE AEQLKKQIAD ARKACADITL AELVSGLEVV GRVQMRTRRT LRGHLAKIYAMHWATDSKLL VSASQDGKLI VWDTYTTNKV HAIPLRSSWV MTCAYAPSGN FVACGGLDNMCSIYSLKSRE GNVKVSRELS AHTGYLSCCR FLDDNNIVTS SGDTTCALWD IETGQQKTVFVGHTGDCMSL AVSPDYKLFI SGACDASAKL WDVREGTCRQ TFTGHESDIN AICFFPNGEAICTGSDDASC RLFDLRADQE LTAYSHESII CGITSVAFSL SGRLLFAGYD DFNCNVWDSL

KCERVGVLSG HDNRVSCLGV TADGMAVATG SWDSFLKIWN (GENBANK Accession No.AAA62620; SEQ ID NO: 3), wherein the protein interaction site residuesare underlined.

Likewise, these residues are located in the same position in a human Gβhaving the amino acid sequence of:

MSELEQLRQE AEQLRNQIRD ARKACGDSTL TQITAGLDPV GRIQMRTRRT LRGHLAKIYAMHWGTDSRLL VSASQDGKLI IWDSYTTNKV HAIPLRSSWV MTCAYAXSGN FVACGGLDNICSIYSLKTRE GNVRVSRELP GHTGYLSCCR FLDDNQIITS SGDTTCALWD IETGQQTVGFAGHSGDVMSL SLAPNGRTFV SGACDASIKL WDVRDSMCRQ TFIGHESDIN AVAFFPNGYAFTTGSDDATC RLFDLRADQE LLMYSHDNH CGITSVAFSR SGRLLLAGYD DFNCNIWDAMKGDRAGVLAG HDNRVSCLGV TDDGMAVATG SWDSFLKIWN (GENBANK Accession No.AAA35922; SEQ ID N0:4), wherein the protein interaction site residuesare underlined.

An agent which interacts with at least one of these amino acid residuesof the protein interaction site of Gβ can bind via various heterogeneousnon-bonded interactions including, but not limited to van der Waalscontacts {e.g., with methionine or leucine), polar contacts {e.g., withaspartate or asparagine), or both {e.g., with lysine, tryptophan, ortyrosine) to contribute to the energy of binding. In general, it isdesirable that the agent interacts with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12 or 13 of the amino acid residues of the protein interaction site ofG/3 to enhance the specificity of the agent for one or more G proteininteracting proteins and therefore one or more G protein-mediatedsignaling pathways.

Determining whether the agent interacts with at least one amino acidresidue of the protein interaction site of the β subunit can beaccomplished using various in vitro or in vivo assays based on detectingprotein-protein interactions between the Gβγ subunits and other peptidesor proteins known to interact with Gβy subunits {e.g., SIGK peptide, Gasubunit, or downstream proteins). An exemplary in vitro assay has beendisclosed herein. This assay consists of obtaining an isolated Gβycomplex; contacting the Gβγ complex with a test agent in the presence ofa peptide that binds at least one amino acid residue of the proteininteraction site of β subunit, {e.g., a SIGK peptide or SIGK peptidederivative of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQID NO: 12, or SEQ ID NO: 13); and detecting the ability of the agent toinhibit the binding of the peptide to the protein interaction site ofthe β subunit using, for example, an ELISA assay. Other phage displayedpeptides identified in the original screen (Scott, et al. (2001) supra)could also be used.

Alternatively, an in vivo assay can be used to determine whether a testagent interacts with at least one amino acid residue of the proteininteraction site of the β subunit. By way of illustration, a two-hybridassay is contemplated where the test agent is contacted with a cellexpressing Gβγ subunits and a peptide such as SIGK, wherein the βsubunit is fused to, e.g., a DNA-binding domain and the SIGK peptide isfused to an activation domain. When the SIGK peptide is bound to theprotein interaction site of GβY, reporter protein expression is induced.If the test agent disrupts the binding of the SIGK peptide to theprotein interaction site of Gβy, reporter protein expression is blocked.

Additional screens such as well-established computational screens orscreens that detect the activity of G protein-dependent downstreamproteins {e.g., PLC β enzymatic activity) are also contemplated for usein conjunction with the assays disclosed herein.

Test agents, also referred to herein as compounds, which can be screenedin accordance with the methods of the present invention are generallyderived from libraries of agents or compounds. Such libraries cancontain either collections of pure agents or collections of agentmixtures. Examples of pure agents include, but are not limited to,proteins, polypeptides, peptides, nucleic acids, oligonucleotides,carbohydrates, lipids, synthetic or semi-synthetic chemicals, andpurified natural products. Examples of agent mixtures include, but arenot limited to, extracts of prokaryotic or eukaryotic cells and tissues,as well as fermentation broths and cell or tissue culture supernates. Inthe case of agent mixtures, the methods of this invention are not onlyused to identify those crude mixtures that possess the desired activity,but also provide the means to monitor purification of the active agentfrom the mixture for characterization and development as a therapeuticdrug. In particular, the mixture so identified can be sequentiallyfractionated by methods commonly known to those skilled in the art whichcan include, but are not limited to, precipitation, centrifugation,filtration, ultrafiltration, selective digestion, extraction,chromatography, electrophoresis or complex formation. Each resultingsubfraction can be assayed for the desired activity using the originalassay until a pure, biologically active agent is obtained. Libraryscreening can be performed as exemplified herein or can be performed inany format that allows rapid preparation and processing of multiplereactions. Stock solutions of the test agents as well as assaycomponents are prepared manually and all subsequent pipeting, diluting,mixing, washing, incubating, sample readout and data collecting is doneusing commercially available robotic pipeting equipment, automated workstations, and analytical instruments for detecting the signal generatedby the assay. Examples of such detectors include, but are not limitedto, luminometers, spectrophotometers, and fluorimeters, and devices thatmeasure the decay of radioisotopes.

To further evaluate the efficacy of a compound identified using ascreening method of the invention, one of skill will appreciate that amodel system of any particular disease or disorder involving G proteinsignaling can be utilized to evaluate the adsorption, distribution,metabolism and excretion of a compound as well as its potential toxicityin acute, sub-chronic and chronic studies. For example, overexpressionof βy inhibitors in NG108-15/D2 cells and rat primary hippocampalneurons has been shown to block δ-opioid and cannabinoidreceptor-induced PKA Ca translocation and gene expression by preventingβy activation of adenylyl cyclase (Yao, et al. (2003) Proc. Natl. Acad.Sci. USA 100:14379-84). Accordingly, to analyze the efficacy of acompound of the instant invention for treating addiction, NG108-15/D2cells and/or rat primary hippocampal neurons are contacted with saidcompound and the effect on PKA Ca translocation is determined. Compoundswhich block δ-opioid and cannabinoid receptor-induced PKA Catranslocation will be useful in treating addiction. Efficacy ofcompounds of the instant invention for preventing or treating heartfailure can be analyzed in a genetic model of murine-dilatedcardiomyopathy which involves the ablation of a muscle-restricted genethat encodes the muscle LIM protein (MLP^(“7”)) (Arber, et al. 1997)Cell 88:393-403). Using this model, it has been demonstrated that abeta-adrenergic receptor kinase 1 inhibitor, BARK-ct, which binds to βγand blocks βγ-dependent activation of beta-adrenergic receptor kinase 1activity, can enhance cardiac contractility in vivo with or withoutisoproterenol (Koch, et al. (1995) Science 268:1350-3) and restore leftventricular size and function (Rockman, eta 1. (1998) Proc. Natl. Acad.Sci. 95:7000-7005). Similarly, compounds of the instant invention whichblock βγ-dependent activation of beta-adrenergic receptor kinase 1activity will be useful in preventing or treating heart failure.

The effectiveness of compounds of the instant to prevent opioidtolerance can be analyzed in acute (Jiang, et al. (1995) J. Pharmacol.Exp. Ther. 273:680-8) and chronic (Wells, et al. (2001) J. Pharmacol.Exp. Ther. 297:597-605) dependence model systems, wherein mice areinjected intracerebroventricularly with a compound of the instantinvention and tolerance to a select opioid (e.g., morphine) isdetermined. Compounds which decrease the amount of opioid necessary toachieve an analgesic effect will be useful in preventing opioidtolerance.

PLC-β2 and -β3 and PI3Kγ have been shown to be involved in thechemoattractant-mediated signal transduction pathway. Mice deficient inP13Kγ lack neutrophil production of Ptdlns (3, 4, 5) P₃, neutrophilmigration, and production of antibodies containing the λ chain whenimmunized with T cell-independent antigen hydroxylnitrophenyl-FICOLL™(Li, et al. (2000) Science 287:1046-1049). Mice lacking PLC-β2 and -β3are deficient in Ca²⁺ release, superoxide production, and MAC-Iup-regulation in neutrophils (Li, et al. (2000) supra). Further, PLC-β2deficient mice exhibit enhanced chemotaxis of different leukocytepopulations and are sensitized to bacteria, viruses, and immunecomplexes (Jiang, et al. (1997) Proc. Natl. Acad. Sci. USA 94 (15):7971-5).

Accordingly, to analyze the efficacy of a compound of the instantinvention for modulating an inflammatory response, mice can beadministered said compound and the effect on neutrophil production ofPtdlns (3, 4, 5) P₃, neutrophil migration, Ca²⁺ efflux, superoxideproduction, production of antibodies containing the λ chain whenimmunized with T cell-independent antigen hydroxylnitrophenyl-FICOLL™ isdetermined. Compounds which selectively potentiate PLC-β2 and -β3 and/orblock PI3Kγ activation thereby inhibiting production of Ptdlns (3, 4, 5)P₃, neutrophil migration, and production of TI-IGλ_(L), will be usefulin treating inflammatory conditions such as arthritis, allergies,Chrohn's Disease and the like. Compounds which selectively block, e.g.,PLC-β2 activation thereby facilitating neutrophil migration will beuseful in facilitating immune responses to bacterial and viralinfections.

Using the screening method of the present invention, various compoundshave now been identified which bind to the protein interaction site of aGβ subunit to interfere with or potentiate physiologically relevantprotein interactions {e.g., Ga subunit and PLC β interactions) therebymodulating the activity of G protein signaling pathways.

1. Compounds and Small Molecules

The following chemical hierarchy is used throughout the specification todescribe and enable the scope of the present invention and toparticularly point out and distinctly claim the units which comprise thecompounds of the present invention, however, unless otherwisespecifically defined, the terms used herein are the same as those of theartisan of ordinary skill. The term “hydrocarbyl” stands for any carbonatom-based unit (organic molecule), said units optionally containing oneor more organic functional group, including inorganic atom comprisingsalts, inter alia, carboxylate salts, quaternary ammonium salts. Withinthe broad meaning of the term “hydrocarbyl” are the classes “acyclichydrocarbyl” and “cyclic hydrocarbyl” which terms are used to dividehydrocarbyl units into cyclic and non-cyclic classes.

As it relates to the following definitions, “cyclic hydrocarbyl” unitsmay comprise only carbon atoms in the ring (carbocyclic and aryl rings)or may comprise one or more heteroatoms in the ring (heterocyclic andheteroaryl). For “carbocyclic” rings the lowest number of carbon atomsin a ring are 3 carbon atoms; cyclopropyl. For “aryl” rings the lowestnumber of carbon atoms in a ring are 6 carbon atoms; phenyl. For“heterocyclic” rings the lowest number of carbon atoms in a ring is 1carbon atom; diazirinyl. Ethylene oxide comprises 2 carbon atoms and isa C₂ heterocycle. For “heteroaryl” rings the lowest number of carbonatoms in a ring is 1 carbon atom; 1,2,3,4-tetrazolyl. The following is anon-limiting description of the terms “acyclic hydrocarbyl” and “cyclichydrocarbyl” as used herein.

A. Substituted and Unsubstituted Acyclic Hydrocarbyl:

-   -   For the purposes of the present invention the term “substituted        and unsubstituted acyclic hydrocarbyl” encompasses 3 categories        of units:

-   1) linear or branched alkyl, non-limiting examples of which include,    methyl (C₁), ethyl (C₂), n-propyl (C₃), iso-propyl (C₃), n-butyl    (C₄), sec-butyl (C₄), iso-butyl (C₄), tert-butyl (C₄), and the like;    substituted linear or branched alkyl, non-limiting examples of which    includes, hydroxymethyl (C₁), chloromethyl (C₁), trifluoromethyl    (C₁), aminomethyl (C₁), 1-chloroethyl (C₂), 2-hydroxyethyl (C₂),    1,2-difluoroethyl (C₂), 3-carboxypropyl (C₃), and the like.

-   2) linear or branched alkenyl, non-limiting examples of which    include, ethenyl (C₂), 3-propenyl (C₃), 1-propenyl (also    2-methylethenyl) (C₃), isopropenyl (also 2-methylethen-2-yl) (C₃),    buten-4-yl (C₄), and the like; substituted linear or branched    alkenyl, non-limiting examples of which include, 2-chloroethenyl    (also 2-chlorovinyl) (C₂), 4-hydroxybuten-1-yl (C₄),    7-hydroxy-7-methyloct-4-en-2-yl (C₉),    7-hydroxy-7-methyloct-3,5-dien-2-yl (C₉), and the like.

-   3) linear or branched alkynyl, non-limiting examples of which    include, ethynyl (C₂), prop-2-ynyl (also propargyl) (C₃),    propyn-1-yl (C₃), and 2-methyl-hex-4-yn-1-yl (C₇); substituted    linear or branched alkynyl, non-limiting examples of which include,    5-hydroxy-5-methylhex-3-ynyl (C₇), 6-hydroxy-6-methylhept-3-yn-2-yl    (C₈), 5-hydroxy-5-ethylhept-3-ynyl (C₉), and the like.    B. Substituted and Unsubstituted Cyclic Hydrocarbyl:    -   For the purposes of the present invention the term “substituted        and unsubstituted cyclic hydrocarbyl” encompasses 5 categories        of units:

-   1) The term “carbocyclic” is defined herein as “encompassing rings    comprising from 3 to 20 carbon atoms, wherein the atoms which    comprise said rings are limited to carbon atoms, and further each    ring can be independently substituted with one or more moieties    capable of replacing one or more hydrogen atoms.” The following are    non-limiting examples of “substituted and unsubstituted carbocyclic    rings” which encompass the following categories of units:    -   i) carbocyclic rings having a single substituted or        unsubstituted hydrocarbon ring, non-limiting examples of which        include, cyclopropyl (C₃), 2-methyl-cyclopropyl (C₃),        cyclopropenyl (C₃), cyclobutyl (C₄), 2,3-dihydroxycyclobutyl        (C₄), cyclobutenyl (C₄), cyclopentyl (C₅), cyclopentenyl (C₅),        cyclopentadienyl (C₅), cyclohexyl (C₆), cyclohexenyl (C₆),        cycloheptyl (C₇), cyclooctanyl (C₈), decalinyl (C₁₀),        2,5-dimethylcyclopentyl (C₅), 3,5-dichlorocyclohexyl (C₆),        4-hydroxycyclohexyl (C₆), and 3,3,5-trimethylcyclohex-1-yl (C₆).    -   ii) carbocyclic rings having two or more substituted or        unsubstituted fused hydrocarbon rings, non-limiting examples of        which include, octahydropentalenyl (C₈), octahydro-1H-indenyl        (C₉), 3a,4,5,6,7,7a-hexahydro-3H-inden-4-yl (C₉),        decahydroazulenyl (C₁₀).    -   iii) carbocyclic rings which are substituted or unsubstituted        bicyclic hydrocarbon rings, non-limiting examples of which        include, bicyclo-[2.1.1]hexanyl, bicyclo[2.2.1]heptanyl,        bicyclo[3.1.1]heptanyl, 1,3-dimethyl[2.2.1]heptan-2-yl,        bicyclo[2.2.2]octanyl, and bicyclo[3.3.3]undecanyl.

-   2) The term “aryl” is defined herein as “units encompassing at least    one phenyl or naphthyl ring and wherein there are no heteroaryl or    heterocyclic rings fused to the phenyl or naphthyl ring and further    each ring can be independently substituted with one or more moieties    capable of replacing one or more hydrogen atoms.” The following are    non-limiting examples of “substituted and unsubstituted aryl rings”    which encompass the following categories of units:    -   i) C₆ or C₁₀ substituted or unsubstituted aryl rings; phenyl and        naphthyl rings whether substituted or unsubstituted,        non-limiting examples of which include, phenyl (C₆),        naphthylen-1-yl (C₁₀), naphthylen-2-yl (CO, 4-fluorophenyl (C₆),        2-hydroxyphenyl (C₆), 3-methylphenyl (C₆),        2-amino-4-fluorophenyl (C₆), 2-(N,N-diethylamino)phenyl (C₆),        2-cyanophenyl (C₆), 2,6-di-tert-butylphenyl (C₆),        3-methoxyphenyl (C₆), 8-hydroxynaphthylen-2-yl (C₁₀),        4,5-dimethoxynaphthylen-1-yl (C₁₀), and 6-cyano-naphthylen-1-yl        (C₁₀).    -   ii) C₆ or C₁₀ aryl rings fused with 1 or 2 saturated rings        non-limiting examples of which include,        bicyclo[4.2.0]octa-1,3,5-trienyl (C₈), and indanyl (C₉).

-   3) The terms “heterocyclic” and/or “heterocycle” are defined herein    as “units comprising one or more rings having from 3 to 20 atoms    wherein at least one atom in at least one ring is a heteroatom    chosen from nitrogen (N), oxygen (O), or sulfur (S), or mixtures of    N, O, and S, and wherein further the ring which comprises the    heteroatom is also not an aromatic ring.” The following are    non-limiting examples of “substituted and unsubstituted heterocyclic    rings” which encompass the following categories of units:    -   i) heterocyclic units having a single ring containing one or        more heteroatoms, non-limiting examples of which include,        diazirinyl (C₁), aziridinyl (C₂), urazolyl (C₂), azetidinyl        (C₃), pyrazolidinyl (C₃), imidazolidinyl (C₃), oxazolidinyl        (C₃), isoxazolinyl (C₃), isoxazolyl (C₃), thiazolidinyl (C₃),        isothiazolyl (C₃), isothiazolinyl (C₃), oxathiazolidinonyl (C₃),        oxazolidinonyl (C₃), hydantoinyl (C₃), tetrahydrofuranyl (C₄),        pyrrolidinyl (C₄), morpholinyl (C₄), piperazinyl (C₄),        piperidinyl (C₄), dihydropyranyl (C₅), tetrahydropyranyl (C₅),        piperidin-2-onyl (valerolactam) (C₅),        2,3,4,5-tetrahydro-1H-azepinyl (C₆), 2,3-dihydro-1H-indole (C₈),        and 1,2,3,4-tetrahydro-quinoline (C₉).    -   ii) heterocyclic units having 2 or more rings one of which is a        heterocyclic ring, non-limiting examples of which include        hexahydro-1H-pyrrolizinyl (C₇),        3a,4,5,6,7,7a-hexahydro-1H-benzo[d]imidazolyl (C₇),        3a,4,5,6,7,7a-hexahydro-1H-indolyl (C₈),        1,2,3,4-tetrahydroquinolinyl (C₉), and        decahydro-1H-cycloocta[b]pyrrolyl (C₁₀).

-   4) The term “heteroaryl” is defined herein as “encompassing one or    more rings comprising from 5 to 20 atoms wherein at least one atom    in at least one ring is a heteroatom chosen from nitrogen (N),    oxygen (O), or sulfur (S), or mixtures of N, O, and S, and wherein    further at least one of the rings which comprises a heteroatom is an    aromatic ring.” The following are non-limiting examples of    “substituted and unsubstituted heterocyclic rings” which encompass    the following categories of units:    -   i) heteroaryl rings containing a single ring, non-limiting        examples of which include, 1,2,3,4-tetrazolyl (C₁),        [1,2,3]triazolyl (C₂), [1,2,4]triazolyl (C₂), triazinyl (C₃),        thiazolyl (C₃), 1H-imidazolyl (C₃), oxazolyl (C₃), furanyl (C₄),        thiopheneyl (C₄), pyrimidinyl (C₄), 2-phenylpyrimidinyl (C₄),        pyridinyl (C₅), 3-methylpyridinyl (C₅), and        4-dimethylaminopyridinyl (C₅)    -   ii) heteroaryl rings containing 2 or more fused rings one of        which is a heteroaryl ring, non-limiting examples of which        include: 7H-purinyl (C₅), 9H-purinyl (C₅), 6-amino-9H-purinyl        (C₅), 5H-pyrrolo[3,2-d]pyrimidinyl (C₆),        7H-pyrrolo[2,3-d]pyrimidinyl (C₆), pyrido[2,3-d]pyrimidinyl        (C₇), 2-phenylbenzo[d]thiazolyl (C₇), 1H-indolyl (C₈),        4,5,6,7-tetrahydro-1-H-indolyl (C₈), quinoxalinyl (C₈),        5-methylquinoxalinyl (C₈), quinazolinyl (C₈), quinolinyl (C₉),        8-hydroxy-quinolinyl (C₉), and isoquinolinyl (C₉).

-   5) C₁-C₆ tethered cyclic hydrocarbyl units (whether carbocyclic    units, C₆ or C₁₀ aryl units, heterocyclic units, or heteroaryl    units) which connected to another moiety, unit, or core of the    molecule by way of a C₁-C₆ alkylene unit. Non-limiting examples of    tethered cyclic hydrocarbyl units include benzyl C₁-(C₆) having the    formula:

-   -   wherein R^(a) is optionally one or more independently chosen        substitutions for hydrogen. Further examples include other aryl        units, inter alia, (2-hydroxyphenyl)hexyl C₆-(C₆);        naphthalen-2-ylmethyl C₁-(C₁₀), 4-fluorobenzyl C₁-(C₆),        2-(3-hydroxy-phenyl)ethyl C₂-(C₆), as well as substituted and        unsubstituted C₃-C₁₀ alkylenecarbocyclic units, for example,        cyclopropylmethyl C₁-(C₃), cyclopentylethyl C₂-(C₅),        cyclohexylmethyl C₁-(C₆). Included within this category are        substituted and unsubstituted C₁-C₁₀ alkylene-heteroaryl units,        for example a 2-picolyl C₁-(C₆) unit having the formula:

-   -   wherein R^(a) is the same as defined above. In addition, C₁-C₁₂        tethered cyclic hydrocarbyl units include C₁-C₁₀        alkyleneheterocyclic units and alkylene-heteroaryl units,        non-limiting examples of which include, aziridinylmethyl C₁-(C₂)        and oxazol-2-ylmethyl C₁-(C₃).

For the purposes of the present invention carbocyclic rings are from C₃to C₂₀; aryl rings are C₆ or C₁₀; heterocyclic rings are from C₁ to C₉;and heteroaryl rings are from C₁ to C₉.

For the purposes of the present invention, and to provide consistency indefining the present invention, fused ring units, as well as spirocyclicrings, bicyclic rings and the like, which comprise a single heteroatomwill be characterized and referred to herein as being encompassed by thecyclic family corresponding to the heteroatom containing ring, althoughthe artisan may have alternative characterizations. For example,1,2,3,4-tetrahydroquinoline having the formula:

is, for the purposes of the present invention, considered a heterocyclicunit. 6,7-Dihydro-5H-cyclopentapyrimidine having the formula:

is, for the purposes of the present invention, considered a heteroarylunit. When a fused ring unit contains heteroatoms in both a saturatedring (heterocyclic ring) and an aryl ring (heteroaryl ring), the arylring will predominate and determine the type of category to which thering is assigned herein for the purposes of describing the invention.For example, 1,2,3,4-tetrahydro-[1,8]naphthyridine having the formula:

is, for the purposes of the present invention, considered a heteroarylunit.

The term “substituted” is used throughout the specification. The term“substituted” is applied to the units described herein as “substitutedunit or moiety is a hydrocarbyl unit or moiety, whether acyclic orcyclic, which has one or more hydrogen atoms replaced by a substituentor several substituents as defined herein below.” The units, whensubstituting for hydrogen atoms are capable of replacing one hydrogenatom, two hydrogen atoms, or three hydrogen atoms of a hydrocarbylmoiety at a time. In addition, these substituents can replace twohydrogen atoms on two adjacent carbons to form said substituent, newmoiety, or unit. For example, a substituted unit that requires a singlehydrogen atom replacement includes halogen, hydroxyl, and the like. Atwo hydrogen atom replacement includes carbonyl, oximino, and the like.A two hydrogen atom replacement from adjacent carbon atoms includesepoxy, and the like. Three hydrogen replacement includes cyano, and thelike. The term substituted is used throughout the present specificationto indicate that a hydrocarbyl moiety, inter alia, aromatic ring, alkylchain; can have one or more of the hydrogen atoms replaced by asubstituent. When a moiety is described as “substituted” any number ofthe hydrogen atoms may be replaced. For example, 4-hydroxyphenyl is a“substituted aromatic carbocyclic ring (aryl ring)”,(N,N-dimethyl-5-amino)octanyl is a “substituted C₈ linear alkyl unit,3-guanidinopropyl is a “substituted C₃ linear alkyl unit,” and2-carboxypyridinyl is a “substituted heteroaryl unit.”

The following are non-limiting examples of units which can substitutefor hydrogen atoms on a carbocyclic, aryl, heterocyclic, or heteroarylunit:

-   -   i) C₁-C₄ linear or branched alkyl; for example, methyl (C₁),        ethyl (C₂), n-propyl (C₃), iso-propyl (C₃), n-butyl (C₄),        iso-butyl (C₄), sec-butyl (C₄), and tert-butyl (C₄);    -   ii) C₆ and C₁₀ substituted or unsubstituted aryl; for example,        4-aminophenyl, 2-sulfamoylphenyl, 3-fluorophenyl, and        2-sulfamoyl-4-aminophenyl;    -   iii) —OR¹⁰⁰; for example, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂CH₃;    -   iv) —C(O)R¹⁰⁰; for example, —COCH₃, —COCH₂CH₃, —COCH₂CH₂CH₃;    -   v) —C(O)OR¹⁰⁰; for example, —CO₂CH₃, —CO₂CH₂CH₃, —CO₂CH₂CH₂CH₃;    -   vi) —C(O)N(R¹⁰⁰)₂; for example, —CONH₂, —CONHCH₃, —CON(CH₃)₂;    -   vii) —N(R¹⁰⁰)₂; for example, —NH₂, —NHCH₃, —N(CH₃)₂,        —NH(CH₂CH₃);    -   viii) halogen: —F, —Cl, —Br, and —I;    -   ix) —CH_(m′)X_(n′); wherein X is halogen, m′ is from 0 to 2,        m′+n′=3; for example, —CH₂F, —CHF₂, —CF₃, —CCl₃, or —CBr₃; and    -   x) —SO₂R¹⁰⁰; for example, —SO₂H; —SO₂CH₃; —SO₂C₆H₅; —SO₂NH₂;        wherein each R¹⁰⁰ is independently hydrogen, substituted or        unsubstituted C₁-C₄ linear, branched, or cyclic alkyl, amino,        alkylamino; or two R¹⁰⁰ units can be taken together to form a        ring comprising 3-7 atoms. Substituents suitable for replacement        of a hydrogen atom are further defined herein below in the        iterations and examples. Substituents suitable for replacement        of a hydrogen atom are further defined herein below.

The present disclosure relates to compounds capable of interacting withat least one amino acid residue of the protein interaction site of the βsubunit of a G protein, and in the broadest sense, the compounds of thepresent disclosure comprise:

-   -   i) 2 or 3 fused rings, the rings comprising from 6 to 16 carbon        atoms and from 0 to 10 heteroatoms chosen from oxygen, nitrogen,        and sulfur; or    -   ii) a first ring system containing 2 or 3 fused rings linked by        a linking group comprising from 1 to 30 atoms to a second ring        system containing 2 or 3 fused rings, the first ring system and        the second ring system each independently containing from 6 to        l6 carbon atoms and from 0 to 10 heteroatoms chosen from oxygen,        nitrogen, and sulfur;

wherein any ring can optionally have one or more hydrogen atomssubstituted by alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkylene,amino, substituted amino, halogen, cyano, nitro, and suflo; or anyhydrogen on a ring can be substituted by a heterocyclic, heteroaryl,cycloalkyl, or aryl ring, the rings that substitute for hydrogen can befurther substituted by alkyl, alkoxy, alkenyl, alkynyl, aryl,arylalkylene, amino, substituted amino, halogen, cyano, nitro, andsuflo;

wherein further two substitutes on a carbon atom can be taken togetherto form a spirocyclic ring having from 3 to 10 carbon atoms and from 0to 3 heteroatoms the spirocyclic ring having one or more hydrogen atomssubstituted by alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkylene,amino, substituted amino, halogen, cyano, nitro, and suflo;

two substituents on a carbon atom can be taken together to form anexocyclic double bond to a unit having from 3 to 10 carbon atoms andfrom 0 to 3 heteroatoms, the unit can also be substituted by alkoxy,alkenyl, alkynyl, aryl, arylalkylene, amino, substituted amino, halogen,cyano, nitro, and suflo.

One aspect of the compounds disclosed herein is represented by theformula:

wherein X¹, X², X³ and X⁴ are ring units each of which may be present orabsent and, therefore, depending upon the selection of units X¹, X², X³and X⁴ and/or their presence or absence in the ring systems, thecompounds of the present disclosure can be any one of the followingnon-limiting examples of ring systems:Systems having 2 core rings:

-   i) fused rings wherein one of the rings is non-aryl,    non-heterocyclic or non-heteroaryl, for example,

-   ii) C₁₀ aryl (naphthylene ring system);-   iii) fused heterocyclic or heteroaryl rings wherein one ring    contains at least one heteroatom, for example,

-   iv) fused rings wherein one ring may comprise a carbonyl or    exocyclic double bond; for example,

Systems having 3 core rings:

-   i) fused ring systems wherein at least two of the rings are    non-aryl, non-heterocyclic or non-heteroaryl, for example,

-   ii) C₁₄ aryl; anthracene, phenanthrene, and 3a¹H-phenalene.-   iii) fused ring systems wherein two of the rings are aryl and the    third ring is cycloalkyl, heterocyclic or heteroaryl, for example,

-   iv) fused rings wherein one ring may comprise a carbonyl or    exocyclic double bond; for example,

The following is a description of the compounds that comprise thepresent disclosure.

The formula:

can be used to describe the compounds disclosed herein.

X¹ and X⁴ are each independently:

-   i) —[C(R^(5a))(R^(5b))]—; for example, —[CH₂]—, —[CH(CH₃)]—,    —[CH(substituted aryl)]—;-   ii) —[CR^(5c)]═; for example, —[CH]═, —[C(substituted aryl)]═;-   iii) —[C(Y)]—; for example, —[C(O)]—, —[C(S)]—, —[C(═NH)]—;-   iv) —[N(R⁶)]—; for example, —[N(H)]—, —[N(substituted aryl)]—;-   v) —[N]═;-   vi) —[O]—;-   vii) —[S]—;

R^(5a), R^(5b), and R^(5c) are each independently chosen from:

-   i) —H;-   ii) C₁-C₁₂ substituted or unsubstituted linear, branched, or cyclic    alkyl; for example, methyl (C₁), ethyl (C₂), n-propyl (C₃),    iso-propyl (C₃), n-butyl (C₄), sec-butyl (C₄), iso-butyl (C₄),    tert-butyl (C₄), n-pentyl (C₅), 1-methylbutyl (C₅), 2-methyl-butyl    (C₅), 3-methylbutyl (C₅), 1,2 dimethylpropyl (C₅),    1,1-dimethylpropyl (C₅), 2,2-dimethylpropyl (C₅), n-hexyl (C₆),    1-methylpentyl (C₆), 2-methylpentyl (C₆), 3-methylpentyl (C₆),    4-methylpentyl (C₆), 1,1-methylbutyl (C₆), 2,2-methylbutyl (C₆),    3,3-methylbutyl (C₆), 1,2-methylbutyl (C₆), 1,3-methylbutyl (C₆),    1,1,2-trimethypropyl (C₆), and 1,2,2-trimethypropyl (C₆);-   iii) C₂-C₁₂ substituted or unsubstituted linear, branched, or cyclic    alkenyl; for example, ethenyl (C₂), n-propenyl (C₃), iso-propenyl    (C₃), n-butenyl (C₄), 1-methylpropen-1-yl (C₄), and    1-methylpropen-2-yl;-   iv) C₂-C₁₂ substituted or unsubstituted linear or branched alkynyl;    prop-1-yn-1-yl (C₃), prop-2-yn-1-yl (propargyl) (C₃);-   v) C₆ or C₁₀ substituted or unsubstituted aryl, for example,    2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 4-methoxyphenyl,    2,3-dimethoxyphenyl, 2,4-dimethoxyphenyl, 3-aminophenyl,    4-aminophenyl, and the like;-   vi) C₇-C₁₅ substituted or unsubstituted arylalkylene; for example,    benzyl, 2-naphthylmethyl (naphthylen-2-ylmethyl);-   vii) C₁-C₉ substituted or unsubstituted heterocyclic;-   viii) C₁-C₁₁ substituted or unsubstituted heteroaryl;-   ix) halogen; fluoro, chloro, bromo, and iodo;-   x) —OR⁷;    -   R⁷ is chosen from:    -   a) —H; thereby forming a hydroxy unit;    -   b) C₁-C₁₂ substituted or unsubstituted linear, branched, or        cyclic alkyl; forming alkoxy units, non-limiting examples        include methoxy (C₁), ethoxy (C₂), n-propoxy (C₃), iso-propoxy        (C₃), n-butoxy (C₄), sec-butoxy (C₄), iso-butoxy (C₄), and        tert-butoxy (C₄);    -   c) C₆ or C₁₀ substituted or unsubstituted aryl;    -   d) C₇-C₁₅ substituted or unsubstituted arylalkylene;    -   e) C₁-C₉ substituted or unsubstituted heterocyclic;    -   f) C₁-C₁₁ substituted or unsubstituted heteroaryl;-   xi) —N(R^(8a))(R^(8b));    -   R^(8a) and R^(8b) are each independently chosen from:    -   a) —H; when R^(8a) and R^(8b) equal hydrogen, —NH₂;    -   b) —OR⁹;        -   R⁹ is hydrogen or C₁-C₄ linear alkyl; for example,            —NH(OCH₃);    -   c) C₁-C₁₂ substituted or unsubstituted linear, branched, or        cyclic alkyl; for example, —NHCH₃, —N(CH₃)₂, —NH(CH₂CH₃);    -   d) C₆ or C₁₀ substituted or unsubstituted aryl; for example,        —NH(4-Cl-phenyl), —N(3,5-dihydroxyphenyl)₂;    -   e) C₇-C₁₂ substituted or unsubstituted arylalkylene; for        example, benzylamino;    -   f) C₁-C₉ substituted or unsubstituted heterocyclic;    -   g) C₁-C₁₁ substituted or unsubstituted heteroaryl; or    -   h) R^(8a) and R^(8b) can be taken together to form a substituted        or unsubstituted ring having from 3 to 10 carbon atoms and from        0 to 3 heteroatoms chosen from oxygen, nitrogen, and sulfur; for        example, aziridin-1-yl pyrrolidin-1-yl, piperidin-1-yl,        piperazin-1-yl, and morpholin-4-yl;-   xii) —CN;-   xiii) —NO₂;-   xiv) —SO₂R¹⁰;    -   R¹⁰ is hydrogen, hydroxyl, substituted or unsubstituted C₁-C₄        linear or branched alkyl; substituted or unsubstituted C₆, C₁₀,        or C₁₄ aryl; C₇-C₁₅ arylalkylene; C₁-C₉ substituted or        unsubstituted heterocyclic; or C₁-C₁₁ substituted or        unsubstituted heteroaryl;-   xv) a R^(5a) and R^(5b) on the same carbon atom can be taken    together to form a substituted or unsubstituted spirocyclic ring    having from 3 to 10 carbon atoms and from 0 to 3 heteroatoms chosen    from oxygen, nitrogen, and sulfur; for example a compound having the    formula:

or

-   xvi) R^(5a) and R^(5b) on the same carbon atom can be taken together    to form an exocyclic double bond having the formula    ═C(R^(5a′))(R^(5b′)), for example,

-   -   wherein R^(5a′) and R^(5b′) are each independently:    -   a) —H;    -   b) C₁-C₁₂ substituted or unsubstituted linear, branched, or        cyclic alkyl;    -   d) C₆ or C₁₀ substituted or unsubstituted aryl;    -   e) C₇-C₁₅ substituted or unsubstituted arylalkylene;    -   r) C₁-C₉ substituted or unsubstituted heterocyclic;    -   g) C₁-C₁₁ substituted or unsubstituted heteroaryl; or    -   h) R^(5a′) and R^(5b′) can be taken together to form a        substituted or unsubstituted ring having from 3 to 10 carbon        atoms and from 0 to 3 heteroatoms chosen from oxygen, nitrogen,        and sulfur.

Each R⁶ is independently chosen from

-   i) —H;-   ii) C₁-C₁₂ substituted or unsubstituted linear, branched, or cyclic    alkyl;-   iii) C₆ or C₁₀ substituted or unsubstituted aryl;-   iv) C₇-C₁₅ substituted or unsubstituted arylalkylene;-   v) C₁-C₉ substituted or unsubstituted heterocyclic;-   vi) C₁-C₁₁ substituted or unsubstituted heteroaryl;-   vii) halogen;-   viii) —N(R^(11a))(R^(11b));    -   R^(11a) and R^(11b) are each independently chosen from:    -   a) —H; or    -   b) C₁-C₄ linear or branched alkyl.

Each Y is independently chosen from:

-   i) ═O;-   ii) ═S;-   iii) ═NR¹²;    -   R¹² is chosen from hydrogen, hydroxyl, or C₁-C₁₂ substituted or        unsubstituted linear or branched alkyl;-   iv) ═C(R^(13a))(R^(13b));    -   R^(13a) and R^(13b) are each independently chosen from    -   a) —H;    -   b) C₁-C₁₂ substituted or unsubstituted linear, branched, or        cyclic alkyl;    -   c) C₆ or C₁₀ substituted or unsubstituted aryl;    -   d) R^(13a) and R^(13b) can be taken together to form a        substituted or unsubstituted cycloalkyl, aryl, heterocyclic, or        heteroaryl ring having from 3 to 10 carbon atoms and from 0 to 3        heteroatoms chosen from oxygen, nitrogen, and sulfur.    -   X² and X³ are each independently:-   i) —[C(R^(14a))(R^(14b))]—;-   ii) —[CR^(14c)]═;-   iii) —[C(Y)]—;-   iv) —[N(R¹⁵)]—;-   v) —[N]═;-   vi) —[O]—;-   vii) —[S]—;-   R^(14a), R^(14b), and R^(14c) are each independently chosen from:-   i) —H;-   ii) C₁-C₁₂ substituted or unsubstituted linear, branched, or cyclic    alkyl;-   iii) C₂-C₁₂ substituted or unsubstituted linear, branched, or cyclic    alkenyl;-   iv) C₂-C₁₂ substituted or unsubstituted linear or branched alkynyl;-   v) halogen;-   vi) —OR¹⁶;    -   R¹⁶ is chosen from:    -   a) —H;    -   b) C₁-C₁₂ substituted or unsubstituted linear, branched, or        cyclic alkyl;    -   c) C₆ or C₁₀ substituted or unsubstituted aryl;    -   d) C₇-C₁₂ substituted or unsubstituted arylalkylene;    -   e) C₁-C₉ substituted or unsubstituted heterocyclic;    -   f) C₁-C₁₁ substituted or unsubstituted heteroaryl;-   vii) —N(R^(17a))(R^(17b));    -   R^(17a) and R^(17b) are each independently chosen from:    -   a) —H;    -   b) —OR²¹;        -   R¹⁸ is hydrogen or C₁-C4 linear alkyl;    -   c) C₁-C₁₂ substituted or unsubstituted linear, branched, or        cyclic alkyl;    -   d) C₆ or C₁₀ substituted or unsubstituted aryl;    -   e) C₇-C₁₅ substituted or unsubstituted arylalkylene;    -   f) C₁-C₉ substituted or unsubstituted heterocyclic;    -   g) C₁-C₁₁ substituted or unsubstituted heteroaryl; or    -   h) R^(17a) and R^(17b) can be taken together to form a        substituted or unsubstituted cycloalkyl, aryl, heterocyclic, or        heteroaryl ring having from 3 to 10 carbon atoms and from 0 to 3        heteroatoms chosen from oxygen, nitrogen, and sulfur;-   viii) —CN;-   ix) —NO₂;-   x) —SO₂R¹⁹;    -   R¹⁹ is hydrogen, hydroxyl, substituted or unsubstituted C₁-C₄        linear or branched alkyl; substituted or unsubstituted C₆, C₁₀,        or C₁₄ aryl; C₇-C₁₅ arylalkylene; C₁-C₉ substituted or        unsubstituted heterocyclic; or C₁-C₁₁ substituted or        unsubstituted heteroaryl;-   xi) R^(14a) and R^(14b) can be taken together to form a spirocyclic    ring can be taken together to form a substituted or unsubstituted    ring having from 3 to 10 carbon atoms and from 0 to 3 heteroatoms    chosen from oxygen, nitrogen, and sulfur;    each R¹⁵ is chosen from-   i) —H;-   ii) C₁-C₁₂ substituted or unsubstituted linear, branched, or cyclic    alkyl;-   iii) C₆ or C₁₀ substituted or unsubstituted aryl;-   iv) C₇-C₁₅ substituted or unsubstituted arylalkylene;-   v) C₁-C₉ substituted or unsubstituted heterocyclic;-   vi) C₁-C₁₁ substituted or unsubstituted heteroaryl;-   vii) halogen;-   viii) —N(R^(20a))(R^(20b));    -   R^(20a) and R^(20b) are each independently chosen from:    -   a) —H; or    -   b) C₁-C₄ linear or branched alkyl;-   each Y is chosen from:-   i) ═O;-   ii) ═S;-   iii) ═NR²¹;    -   R²¹ is chosen from hydrogen, hydroxyl, or C₁-C₁₂ substituted or        unsubstituted linear or branched alkyl;-   iv) ═C(R^(22a))(R^(22b));    -   R^(22a) and R^(22b) are each independently chosen from    -   a) —H;    -   b) C₁-C₁₂ substituted or unsubstituted linear, branched, or        cyclic alkyl;    -   c) C₆ or C₁₀ substituted or unsubstituted aryl;    -   d) R^(22a) and R^(22b) can be taken together to form a        substituted or unsubstituted cycloalkyl, aryl, heterocyclic, or        heteroaryl ring having from 3 to 10 carbon atoms and from 0 to 3        heteroatoms chosen from oxygen, nitrogen, and sulfur;)        when X² an X³ are each —[C(R^(14a))(R^(14b))]— or are each        —[CR^(14c)]═, and R^(14a) or an R^(14c) unit from X² and X³ can        be taken together to form a substituted or unsubstituted        cycloalkyl, aryl, heterocyclic or heteroaryl ring containing        from 4 to 10 carbon atoms any of which carbon atoms can be a        carbonyl carbon, and from 0 to 3 heteroatoms chosen from oxygen,        nitrogen, and sulfur; wherein the rings formed from an R^(14a)        or R^(14b) on X² and an R^(14a) or R^(14b) on X³ cam be        substituted by one or more units R^(1′), R^(2′), R^(3′), or R⁴,        wherein each R^(1′), R^(2′), R^(3′), or R⁴, can be the same as        defined herein below for R¹, R², R³, and R⁴. Non-limiting        examples of rings comprising R¹, R², R³, R⁴, R^(1′), R^(2′),        R^(3′), or R⁴, units include, for example, rings having the        formulae:

-   -   R¹, R², R³, and R⁴ are each independently chosen from:

-   i) —H;

-   ii) C₁-C₁₂ substituted or unsubstituted linear, branched, or cyclic    alkyl;

-   iii) C₂-C₁₂ substituted or unsubstituted linear, branched, or cyclic    alkenyl;

-   iv) C₂-C₁₂ substituted or unsubstituted linear or branched alkynyl;

-   v) C₆ or C₁₀ substituted or unsubstituted aryl;

-   vi) C₇-C₁₅ substituted or unsubstituted arylalkylene;

-   vii) C₁-C₉ substituted or unsubstituted heterocyclic;

-   viii) C ₁-C ₁₁ substituted or unsubstituted heteroaryl;

-   ix) —OR²⁷;    -   R²⁷ is chosen from:    -   a) —H;    -   b) C₁-C₁₂ substituted or unsubstituted linear, branched, or        cyclic alkyl;    -   c)₆ or C₁₀ substituted or unsubstituted aryl;    -   d) C₁-C₉ substituted or unsubstituted heterocyclic;    -   e) C ₁-C ₁₁ substituted or unsubstituted heteroaryl;

-   x) —N(R^(28a))(R^(28b));    -   R^(28a) and R^(28b) are each independently chosen from:    -   a) —H;    -   b) —OR²⁹;        -   R²⁹ is hydrogen or C₁-C4 linear alkyl;    -   c) C₁-C₁₂ substituted or unsubstituted linear, branched, or        cyclic alkyl;    -   d) C₆ or C₁₀ substituted or unsubstituted aryl;    -   e) C₇-C₁₅ substituted or unsubstituted arylalkylene;    -   f) C₁-C₉ substituted or unsubstituted heterocyclic;    -   g) C₁-C₁₁ substituted or unsubstituted heteroaryl; or    -   h) R^(28a) and R^(28b) can be taken together to form a        substituted or unsubstituted cycloalkyl, aryl, heterocyclic, or        heteroaryl ring having from 3 to 10 carbon atoms and from 0 to 3        heteroatoms chosen from oxygen, nitrogen, and sulfur;

-   xi) halogen;

-   xii) —CN;

-   xiii) —NO₂;

-   xiv) —SO₂R³⁰;    -   R³⁰ is hydrogen, hydroxyl, substituted or unsubstituted C₁-C₄        linear or branched alkyl; substituted or unsubstituted C₆, C₁₀,        or C₁₄ aryl; C₇-C₁₅ arylalkylene; C₁-C₉ substituted or        unsubstituted heterocyclic; or C₁-C₁₁ substituted or        unsubstituted heteroaryl;        a R^(5a) unit from X¹ and a R^(14a) unit from X² or a R^(5a)        unit from X⁴ and a R^(14a) unit from X³ can be taken together to        form a substituted or unsubstituted cycloalkyl, aryl,        heterocyclic, or heteroaryl ring having from 3 to 10 carbon        atoms and from 0 to 3 heteroatoms chosen from oxygen, nitrogen,        and sulfur.

In addition to units comprising one 2 or 3 fused ring system, R¹, R²,R³, R⁴, X¹, X², X³, and X⁴ are each independently a unit containing alinking group L capable of linking a first ring system with a secondring system through any of the R¹, R², R³, R⁴, X¹, X², X³, or X⁴ unitsof each ring, the linked rings having the formula:

L is a linking unit having the formula:—[C(R^(23a))(R^(23b))]_(j)[W]_(k)[C(R^(24a))(R^(24b))]_(m)[Z]_(n)[C(R^(25a))(R^(25b))]_(o)—

-   R^(23a), R^(23b), R^(24a), R^(24b), R^(25a), and R^(25b) are each    independently chosen from;-   i) —H; or-   ii) C₁-C₄ substituted or unsubstituted linear or branched alkyl;-   iii) C₆ substituted or unsubstituted aryl; or-   iv) C₇-C₁₂ substituted or unsubstituted arylalkylene;-   W and Z are each independently chosen from:-   i) -M-;-   ii) —C(=M)-;-   iii) —C(=M)M-;-   iv) -MC(=M)-;-   v) -MC(=M)M-;-   vi) -MC(=M)C(=M)M-;-   vii) -MC(=M)MC(=M)M-;    each M is independently chosen from O, S, and NR²⁶; R²⁶ is hydrogen,    hydroxyl, or C₁-C₄ linear or branched alkyl;    the indices k and n are 0 or 1; the indices j, m, and o are from 0    to 12.

As described herein above, the compounds presently disclosed cancomprise substituted or unsubstituted heterocyclic and heteroaryl rings.

The following are non-limiting examples of substituted or unsubstitutedC₂, C₃, C₄ 5-member heterocyclic rings, wherein R^(a) represents one ormore substitutions for hydrogen when the substitution is present.

-   -   i) a substituted pyrrolidinyl ring having the formula:

-   -   ii) a substituted pyrrolyl ring having the formula:

-   -   iii) a substituted 4,5-dihydroimidazolyl ring having the        formula:

-   -   iv) a substituted pyrazolyl ring having the formula:

-   -   v) an substituted imidazolyl ring having the formula:

-   -   vi) a substituted [1,2,3]triazolyl ring having the formula:

-   -   vii) a substituted [1,2,4] triazolyl ring having the formula:

-   -   viii) a substituted tetrazolyl ring having the formula:

-   -   ix) a substituted [1,3,4] or [1,2,4]oxadiazolyl ring having the        formula:

-   -   x) a substituted pyrrolidinonyl ring having the formula:

-   -   xi) a substituted imidazolidinonyl ring having the formula:

-   -   xii) a substituted imidazol-2-only ring having the formula:

-   -   xiii) a substituted oxazolyl ring having the formula:

-   -   xiv) a substituted isoxazolyl ring having the formula:

-   -   xv) a substituted thiazolyl ring having the formula:

-   -   xvi) a substituted furanly ring having the formula:

-   -   xvii) a substituted thiophenyl having the formula:

The following are non-limiting examples of substituted or unsubstitutedC₃, C₄, C₅ 6-member heterocyclic rings, wherein R^(a) represents one ormore substitutions for hydrogen when the substitution is present.

-   -   i) a substituted morpholinyl ring having the formula:

-   -   ii) a substituted piperidinyl ring having the formula:

-   -   iii) a substituted pyridinyl ring having the formula:

-   -   iv) a substituted pyrimidinyl ring having the formula:

-   -   v) a substituted piperazinyl ring having the formula:

-   -    and    -   vi) a substituted triazinyl ring having the formula:

The R^(a) substitutions for hydrogen in the above heterocyclic andheteroaryl units include the following non-limiting examples, eachindependently chosen from:

-   -   i) C₁-C₄ linear or branched alkyl; for example, methyl (C₁),        ethyl (C₂), n-propyl (C₃), iso-propyl (C₃), n-butyl (C₄),        iso-butyl (C₄), sec-butyl (C₄), and tert-butyl (C₄);    -   ii) C₆ and C₁₀ substituted or unsubstituted aryl; for example,        4-aminophenyl, 2-sulfamoylphenyl, 3-fluorophenyl, and        2-sulfamoyl-4-aminophenyl;    -   iii) —OR^(a); for example, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂CH₃;    -   iv) —C(O)R^(a); for example, —COCH₃, —COCH₂CH₃, —COCH₂CH₂CH₃;    -   v) —C(O)OR^(a); for example, —CO₂CH₃, —CO₂CH₂CH₃, —CO₂CH₂CH₂CH₃;    -   vi) —C(O)N(R^(a))₂; for example, —CONH₂, —CONHCH₃, —CON(CH₃)₂;    -   vii) —N(R^(a))₂; for example, —NH₂, —NHCH₃, —N(CH₃)₂,        —NH(CH₂CH₃);    -   viii) halogen: —F, —Cl, —Br, and —I;    -   ix) —CH_(m′)X^(n′); wherein X is halogen, m′ is from 0 to 2,        m′+n′=3; for example, —CH₂F, —CHF₂, —CF₃, —CCl₃, or —CBr₃; and    -   x) —SO₂R^(a0); for example, —SO₂H; —SO₂CH₃; —SO₂C₆H₅; —SO₂NH₂;        wherein each R^(a) is independently hydrogen, substituted or        unsubstituted C₁-C₄ linear, branched, or cyclic alkyl, amino,        alkylamino; or two R^(a) units can be taken together to form a        ring comprising 3-7 atoms. Substituents suitable for replacement        of a hydrogen atom are further defined herein below in the        iterations and examples.

A first category of compounds capable of interacting with at least oneamino acid residue of the protein interaction site of the β subunit of aG protein according to the present disclosure relates to compoundshaving the formula:

wherein X¹ and X⁴ are each —[C(Y)]—, X² and X³ are each —[CR^(14c)]═;and the R^(14c) units from X² and X³ are taken together to form acycloalkyl or aryl ring having from 4 to 10 carbon atoms and R¹, R², R³,and R⁴ are further defined.

A first embodiment of this category of the present disclosure relates tocompounds wherein Y equals (═O) and the R^(14c) units are taken togetherto form an aryl ring, the compounds having the formula:

wherein R¹, R², R³, and R⁴ are each independently chosen from:

-   -   i) —H;    -   ii) C₆ or C₁₀ substituted or unsubstituted aryl;    -   iii) —OR²⁷;        -   R²⁷ is chosen from:        -   a) —H;        -   b) C₁-C₁₂ substituted or unsubstituted linear, branched, or            cyclic alkyl;    -   iv) —N(R^(28a))(R^(28b));        -   R^(28a) and R^(28b) are each independently chosen from:        -   a) —H;        -   d) C₆ or C₁₀ substituted or unsubstituted aryl;    -   v) halogen;    -   vi) —SO₂R³⁰;        -   R³⁰ is hydrogen, substituted or unsubstituted C₁-C₄ linear            or branched alkyl.

The substitutions for hydrogen in the above units are each independentlychosen from:

-   -   i) C₁-C₄ linear or branched alkyl; for example, methyl (C₁),        ethyl (C₂), n-propyl (C₃), iso-propyl (C₃), n-butyl (C₄),        iso-butyl (C₄), sec-butyl (C₄), and tert-butyl (C₄);    -   ii) C₆ and C₁₀ substituted or unsubstituted aryl; for example,        4-aminophenyl, 2-sulfamoylphenyl, 3-fluorophenyl, and        2-sulfamoyl-4-aminophenyl;    -   iii) —OR¹⁰⁰; for example, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂CH₃;    -   iv) —C(O)R¹⁰⁰; for example, —COCH₃, —COCH₂CH₃, —COCH₂CH₂CH₃;    -   v) —C(O)OR¹⁰⁰; for example, —CO₂CH₃, —CO₂CH₂CH₃, —CO₂CH₂CH₂CH₃;    -   vi) —C(O)N(R¹⁰⁰)₂; for example, —CONH₂, —CONHCH₃, —CON(CH₃)₂;    -   vii) —N(R¹⁰⁰)₂; for example, —NH₂, —NHCH₃, —N(CH₃)₂,        —NH(CH₂CH₃);    -   viii) halogen: —F, —Cl, —Br, and —I;    -   ix) —CH_(m′)X_(n′); wherein X is halogen, m′ is from 0 to 2,        m′+n′=3; for example, —CH₂F, —CHF₂, —CF₃, —CCl₃, or —CBr₃; and    -   x) —SO₂R¹⁰⁰; for example, —SO₂H; —SO₂CH₃; —SO₂C₆H₅; —SO₂NH₂;        wherein each R¹⁰⁰ is independently hydrogen, substituted or        unsubstituted C₁-C₄ linear, branched, or cyclic alkyl, amino,        alkylamino; or two R¹⁰⁰ units can be taken together to form a        ring comprising 3-7 atoms. Substituents suitable for replacement        of a hydrogen atom are further defined herein below in the        iterations and examples.

One iteration of this embodiment relates to compounds having theformula:)

wherein R¹ is —N(R^(28a))(R^(28b)), for which R^(28a) and R^(28b) areeach hydrogen; R² is —SO₂R³⁰, R³⁰ is hydrogen; R³ is hydrogen; and R⁴ is—N(R^(28a))(R^(28b)); R^(28a) and R^(28b) are further defined herein forthis embodiment.

One example of this iteration includes R⁴ units wherein both R^(28a) andR^(28b) are hydrogen, thereby providing1,3-diamino-9,10-dioxo-9,10-dihydroanthracene-2-sulphonic acid havingthe formula:

Another example of this iteration includes compounds wherein R^(28b) ishydrogen and R^(28a) is phenyl or substituted phenyl thereby providing1-amino-9,10-dioxo-4-(substituted or unsubstitutedphenylamino)-9,10-dihydroanthracene-2-sulphonic acids having theformula:

wherein R¹⁰⁰ represents one or more substitutions for hydrogen.

Non-limiting examples of the R^(28a) units comprising R⁴ include phenyl,2-sulfophenyl, 3-sulfophenyl, 4-sulfophenyl, 2-aminophenyl,3-aminophenyl, 4-aminophenyl, 2-sulfamoylphenyl, 3-sulfamoylphenyl,4-sulfamoylphenyl, 2-methylaminophenyl, 3-methylaminophenyl,4-methylaminophenyl, 2-(dimethylamino)phenyl, 3-(dimethyl-amino)phenyl,4-(dimethylamino)phenyl, 2-methylphenyl, 3-methylphenyl,4-methyl-phenyl, 2-(hydroxymethyl)phenyl, 3-(hydroxymethyl)phenyl,4-(hydroxymethyl)phenyl, 2-sulfo-4-aminophenyl, 3-sulfo-4-aminophenyl,2-amino-4-sulfophenyl, 3-amino-4-sulfophenyl, and 3-amino-5-sulfophenyl.

Another iteration of this embodiment includes compounds having theformula:

wherein R² is halogen and the R^(28a) and R^(28b) units for R⁴ are thesame as defined herein above, thereby providing1-amino-2-halogen-4-(substituted or unsubstitutedphenylamino)anthracene-9,10-diones.

This iteration includes the following examples of athracene-9,10-diones:

wherein R¹⁰⁰ is the same as described herein above thereby providingnon-limiting examples of R^(28a) that include phenyl, 2-sulfophenyl,3-sulfophenyl, 4-sulfophenyl, 2-aminophenyl, 3-aminophenyl,4-aminophenyl, 2-sulfamoylphenyl, 3-sulfamoyl-phenyl, 4-sulfamoylphenyl,2-methylaminophenyl, 3-methylaminophenyl, 4-methylaminophenyl,2-(dimethylamino)phenyl, 3-(dimethyl-amino)phenyl,4-(dimethylamino)phenyl, 2-methylphenyl, 3-methylphenyl,4-methyl-phenyl, 2-(hydroxymethyl)phenyl, 3-(hydroxymethyl)phenyl,4-(hydroxymethyl)phenyl, 2-sulfo-4-aminophenyl, 3-sulfo-4-aminophenyl,2-amino-4-sulfophenyl, 3-amino-4-sulfophenyl, and 3-amino-5-sulfophenyl.

A further iteration of this embodiment includes compounds having theformula:

wherein R²⁷ is chosen from hydrogen or C₁-C₁₂ substituted orunsubstituted linear, branched, or cyclic alkyl and R^(28a) and R^(28b)are the same as defined herein above, thereby providing1-amino-2-alkoxy-4-(substituted or unsubstitutedphenylamino)anthracene-9,10-diones. Non-limiting examples of R²⁷ includemethyl (C₁), ethyl (C₂), n-propyl (C₃), iso-propyl (C₃), n-butyl (C₄),sec-butyl (C₄), iso-butyl (C₄), and tent-butyl (C₄).

This iteration includes the following examples of athracene-9,10-dioneshaving the formula:

wherein R¹⁰⁰ is the same as described herein above thereby providingnon-limiting examples of R^(28a) that include phenyl, 2-sulfophenyl,3-sulfophenyl, 4-sulfophenyl, 2-aminophenyl, 3-aminophenyl,4-aminophenyl, sulfamoylphenyl, 3-sulfamoyl-phenyl, 4-sulfamoylphenyl,2-methylaminophenyl, 3-methylaminophenyl, 4-methylaminophenyl,2-(dimethylamino)phenyl, 3-(dimethyl-amino)phenyl,4-(dimethylamino)phenyl, 2-methylphenyl, 3-methylphenyl,4-methyl-phenyl, 2-(hydroxymethyl)phenyl, 3-(hydroxymethyl)phenyl,4-(hydroxymethyl)phenyl, 2-sulfo-4-aminophenyl, 3-sulfo-4-aminophenyl,2-amino-4-sulfophenyl, 3-amino-4-sulfophenyl, and 3-amino-5-sulfophenyl.

The following Tables I-IV provide binding data (IC₅₀) for compoundsrepresentative compounds of this embodiment at receptors PLCβ₂, PLCβ₃,and PI₃Kγ.

TABLE 2 Elisa Compound IC₅₀

56 μM

12 μM

19 μM

59 μM

18 μM

TABLE 3 PLCβ₂ Compound IC₅₀

  75 μM

  14 μM

>30 μM

TABLE 4 PLC β₃ Compound IC₅₀

58 μM

12 μM

15 μM

62 μM

TABLE 5 PI₃Kγ Compound IC₅₀

 10 μM

2.5 μM

  6 μM

 16 μM PI₃Kγ= phosphoinsotide 3 kinase γ.

Another category of compounds capable of interacting with at least oneamino acid residue of the protein interaction site of the β subunit of aG protein according to the present disclosure relates to compoundshaving the formula:

wherein X¹ is —[CR^(5c)]═, X⁴ is —[O]—; X² and X³ are each —[CR^(14c)]═;and the R^(14c) unit from X² and X³ are taken together to form anunsaturated cycloalkyl ring having 6 carbon atoms.

-   -   R¹, R², R³, and R⁴ are each independently chosen from:    -   i) —H;    -   ii) C₆ or C₁₀ substituted or unsubstituted aryl; or    -   iii) —OR²⁷;        -   R²⁷ is chosen from:        -   a) —H; or        -   b) C₁-C₁₂ substituted or unsubstituted linear, branched, or            cyclic alkyl.    -   R^(1′), R^(2′), and R^(4′) are each independently chosen from:    -   i) —H;    -   ii) C₆ or C₁₀ substituted or unsubstituted aryl; or    -   iii) —OR²⁷;        -   R²⁷ is chosen from:        -   a) —H; or        -   b) C₁-C₁₂ substituted or unsubstituted linear, branched, or            cyclic alkyl.    -   R^(5c) is chosen from:    -   i) —H;    -   ii) C₁-C₁₂ substituted or unsubstituted linear, branched, or        cyclic alkyl;    -   iii) C₂-C₁₂ substituted or unsubstituted linear, branched, or        cyclic alkenyl; or    -   iv) C₆ or C₁₀ substituted or unsubstituted aryl.

The substitutions for hydrogen in the above units are each independentlychosen from:

-   -   i) C₁-C₄ linear or branched alkyl; for example, methyl (C₁),        ethyl (C₂), n-propyl (C₃), iso-propyl (C₃), n-butyl (C₄),        iso-butyl (C₄), sec-butyl (C₄), and tert-butyl (C₄);    -   ii) C₆ and C₁₀ substituted or unsubstituted aryl; for example,        4-aminophenyl, 2-sulfamoylphenyl, 3-fluorophenyl, and        2-sulfamoyl-4-aminophenyl;    -   iii) —OR¹⁰⁰; for example, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂CH₃;    -   iv) —C(O)R¹⁰⁰; for example, —COCH₃, —COCH₂CH₃, —COCH₂CH₂CH₃;    -   v) —C(O)OR¹⁰⁰; for example, —CO₂CH₃, —CO₂CH₂CH₃, —CO₂CH₂CH₂CH₃;    -   vi) —C(O)N(R¹⁰⁰)₂; for example, —CONH₂, —CONHCH₃, —CON(CH₃)₂;    -   vii) —N(R¹⁰⁰)₂; for example, —NH₂, —NHCH₃, —N(CH₃)₂,        —NH(CH₂CH₃);    -   viii) halogen: —F, —Cl, —Br, and —I;    -   ix) —CH_(m′)X_(n′); wherein X is halogen, m′ is from 0 to 2,        m′+n′=3; for example, —CH₂F, —CHF₂, —CF₃, —CCl₃, or —CBr₃; and    -   x) —SO₂R¹⁰⁰; for example, —SO₂H; —SO₂CH₃; —SO₂C₆H₅; —SO₂NH₂;        wherein each R¹⁰⁰ is independently hydrogen, substituted or        unsubstituted C₁-C₄ linear, branched, or cyclic alkyl, amino,        alkylamino; or two R¹⁰⁰ units can be taken together to form a        ring comprising 3-7 atoms. Substituents suitable for replacement        of a hydrogen atom are further defined herein below in the        iterations and examples.

One category of compounds disclosed herein are the4,5,6-trihydroxy-9-substituted-3H-xanten-3-ones having the formula:

wherein R^(5c) is chosen from:

-   -   i) C₃-C₁₂ substituted or unsubstituted cyclic alkyl;    -   ii) C₃-C₁₂ substituted or unsubstituted cyclic alkenyl;    -   iii) C₆ or C₁₀ substituted or unsubstituted aryl; and    -   iv) C₇-C₁₅ substituted or unsubstituted arylalkylene.

Suitable substitutions for the above R^(5c) are

-   -   i) C₁-C₄ linear or branched alkyl; for example, methyl (C₁),        ethyl (C₂), n-propyl (C₃), iso-propyl (C₃), n-butyl (C₄),        iso-butyl (C₄), sec-butyl (C₄), and tert-butyl (C₄);    -   ii) C₆ and C₁₀ substituted or unsubstituted aryl; for example,        4-aminophenyl, 2-sulfamoylphenyl, 3-fluorophenyl, and        2-sulfamoyl-4-aminophenyl;    -   iii) —OR¹⁰⁰; for example, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂CH₃;    -   iv) —C(O)R¹⁰⁰; for example, —COCH₃, —COCH₂CH₃, —COCH₂CH₂CH₃;    -   v) —C(O)OR¹⁰⁰; for example, —CO₂CH₃, —CO₂CH₂CH₃, —CO₂CH₂CH₂CH₃;    -   vi) —C(O)N(R¹⁰⁰)₂; for example, —CONH₂, —CONHCH₃, —CON(CH₃)₂;    -   vii) —N(R¹⁰⁰)₂; for example, —NH₂, —NHCH₃, —N(CH₃)₂,        —NH(CH₂CH₃);    -   viii) halogen: —F, —Cl, —Br, and —I;    -   ix) —CH_(m′)X_(n′); wherein X is halogen, m′ is from 0 to 2,        m′+n′=3; for example, —CH₂F, —CHF₂, —CF₃, —CCl₃, or —CBr; and    -   x) —SO₂R¹⁰⁰; for example, —SO₂H; —SO₂CH₃; —SO₂C₆H₅; —SO₂NH₂.

However, it has been discovered that there is a need for understandingthe relationship between the core 3H-xanten-3-one ring, the moietiesthat comprise R^(5a) and binding. For example,6-hydroxy-9-methyl-3H-xanthen-3-one having the formula:

was found not to bind to the G protein β subunit. Further addition ofhydroxyl units to the core ring, for example,2,6,7-trihydroxy-9-methyl-3H-xanthen-3-one, having the formula:

did not provide a compound that exhibited binding. Extending the lengthof R^(5a) and allowing for both free rotation of the R^(5a) substituent(alkyl unit) and restricted rotation of the R^(5a) unit (alkenyl unit),as well as providing a unit capable of increased hydrogen bonding(—C(O)OR¹⁰⁰ unit wherein R¹⁰⁰ is hydrogen) provided two compounds,3-(6-hydroxy-3-oxo-3H-xanthen-9-yl)propionic acid and(E)-3-(6-hydroxy-3-oxo-3H-xanthe-9-yl)acrylic acid having the formulaerespectively:

Neither of these compounds showed any binding to the G protein βsubunit.

However, when these two R^(5c) substituents were attached to thepresently disclosed 4,5,6-trihydroxy-9-substituted-3H-xanten-3-one ringsystem, thereby forming the compounds3-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-ylpropanoic acid and(E)-3-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)acrylic acid having theformulae:

both of these compounds elicited significant binding; 2.5 μM and 200 nmrespectively.

One embodiment of this category relates to compounds having the formula:

wherein R^(5c) is a substituted or unsubstituted cycloalkyl unit.Non-limiting examples of unsubstituted cycloalkyl units include:

-   -   i) mono-cyclic alkyl; for example, cyclopropyl (C₃), cyclobutyl        (C₄), cyclopentyl (C₅), cyclohexyl (C₆), cycloheptyl (C₇), and        the like;    -   ii) mono-cyclic alkenyl; for example, cyclopent-1-en-1-yl (C₅),        cyclohex-2-en-1-yl, and the like;    -   iii) bicycloalkyl; for example, bicyclo[3.1.0]hexanyl (C₆),        bicyclo[4.1.0]-heptanyl (C₇), bicyclo[3.1.1]heptanyl (C₇);        bicyclo[4.1.1]octanyl (C₈), and the like;    -   iv) unsaturated bicycloalkyl; for example,        bicyclo[3.1.0]hex-2-enyl (C₆), bicyclo[4.1.0]-hept-3-enyl (C₇),        bicyclo[3.1.1]hept-2-enyl (C₇); bicyclo[4.1.1]oct-3-enyl (C₈),        and the like;    -   v) bridged fused ring alkyl; for example, the following unit,        tricyclo[4.4.1] decalin-2-yl, (C₁₁) numbered as indicated;

One iteration of this embodiment relatest compounds wherein R^(5c) is asubstituted or unsubstituted cycloalkyl ring, for example,

wherein R^(b) represents one or more optionally present substitutionsfor hydrodgen. Non-limiting examples of R^(b) units include unitsindependently chosen from:

-   -   i) C₁-C₄ linear or branched alkyl; for example, methyl (C₁),        ethyl (C₂), n-propyl (C₃), iso-propyl (C₃), n-butyl (C₄),        iso-butyl (C₄), sec-butyl (C₄), and tert-butyl (C₄);    -   ii) C₆ and C₁₀ substituted or unsubstituted aryl; for example,        4-aminophenyl, 2-sulfamoylphenyl, 3-fluorophenyl, and        2-sulfamoyl-4-aminophenyl;    -   iii) —OR²⁰⁰; for example, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂CH₃;    -   iv) —C(O)R²⁰⁰; for example, —COCH₃, —COCH₂CH₃, —COCH₂CH₂CH₃;    -   v) —C(O)OR²⁰⁰; for example, —CO₂CH₃, —CO₂CH₂CH₃, —CO₂CH₂CH₂CH₃;    -   vi) —C(O)N(R²⁰⁰)₂; for example, —CONH₂, —CONHCH₃, —CON(CH₃)₂;    -   vii) —N(R²⁰⁰)₂; for example, —NH₂, —NHCH₃, —N(CH₃)₂,        —NH(CH₂CH₃);    -   viii) halogen: —F, —Cl, —Br, and —I;    -   ix) —CH_(m′)X_(n′); wherein X is halogen, m′ is from 0 to 2,        m′+n′=3; for example, —CH₂F, —CHF₂, —CF₃, —CCl₃, or —CBr₃; and    -   x) —SO₂R²⁰⁰; for example, —SO₂H; —SO₂CH₃; —SO₂C₆H₅; —SO₂NH₂;        wherein each R²⁰⁰ is independently hydrogen, substituted or        unsubstituted C₁-C₄ linear, branched, or cyclic alkyl, amino,        alkylamino; or two R²⁰⁰ units can be taken together to form a        ring comprising 3-7 atoms.

Non-limiting examples of compounds according to this embodiment include2-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)cyclohexanecarboxylic acidhaving the formula:

2-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)cyclohexanecarboxyamide havingthe formula:

9-(2-aminocyclohexyl)-4,5,6-trihydroxy-3H-xathnen-3-one having theformula:

3-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)cyclohexanecarboxylic acidhaving the formula:

4-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)cyclohexanecarboxylic acidhaving the formula:

5-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)cyclohexane-1,3-dicarboxylicacid having the formula:

7-methyl-3-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)bicyclo[2.2.1]hept-5-ene-2-carboxylicacid having the formula:

1,4,5,6,7,7-hexachloro-3-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)bicyclo[2.2.1]hept-5-ene-2-carboxylicacid having the formula:

and7,8,9,10,11,11-hexachloro-3-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)tricyclo[4.4.1]-decaline-2-carboxylicacid having the formula:

Another category relates to compounds having the formula:

wherein R^(5c) is the same as defined herein above, and R¹ and R^(1′)can be hydrogen or a substitution independently chosen from:

-   -   i) C₁-C₄ linear or branched alkyl; for example, methyl (C₁),        ethyl (C₂), n-propyl (C₃), iso-propyl (C₃), n-butyl (C₄),        iso-butyl (C₄), sec-butyl (C₄), and tert-butyl (C₄);    -   ii) C₆ and C₁₀ substituted or unsubstituted aryl; for example,        4-aminophenyl, 2-sulfamoylphenyl, 3-fluorophenyl, and        2-sulfamoyl-4-aminophenyl;    -   iii) —OR²⁰¹; for example, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂CH₃;    -   iv) —C(O)R²⁰¹; for example, —COCH₃, —COCH₂CH₃, —COCH₂CH₂CH₃;    -   v) —C(O)OR²⁰¹; for example, —CO₂CH₃, —CO₂CH₂CH₃, —CO₂CH₂CH₂CH₃;    -   vi) —C(O)N(R²⁰¹)₂; for example, —CONH₂, —CONHCH₃, —CON(CH₃)₂;    -   vii) —N(R²⁰¹)₂; for example, —NH₂, —NHCH₃, —N(CH₃)₂,        —NH(CH₂CH₃);    -   viii) halogen: —F, —Cl, —Br, and —I;    -   ix) —CH_(m′)X_(n′); wherein X is halogen, m′ is from 0 to 2,        m′+n′=3; for example, —CH₂F, —CHF₂, —CF₃, —CCl₃, or —CBr₃; and    -   x) —SO₂R²⁰¹; for example, —SO₂H; —SO₂CH₃; —SO₂C₆H₅; —SO₂NH₂;        wherein each R²⁰¹ is independently hydrogen, substituted or        unsubstituted C₁-C₄ linear, branched, or cyclic alkyl, amino,        alkylamino; or two R²⁰¹ units can be taken together to form a        ring comprising 3-7 atoms. Examples of this iteration include        compound having the formulae:

wherein R^(b) represents one or more optionally present substitutionsfor hydrogen as defined herein above.

Another category relates to compounds having the formula:

wherein R^(5c) is the same as defined herein above, and R² and R^(2′)can be hydrogen or a substitution independently chosen from:

-   -   i) C₁-C₄ linear or branched alkyl; for example, methyl (C₁),        ethyl (C₂), n-propyl (C₃), iso-propyl (C₃), n-butyl (C₄),        iso-butyl (C₄), sec-butyl (C₄), and tert-butyl (C₄);    -   ii) C₆ and C₁₀ substituted or unsubstituted aryl; for example,        4-aminophenyl, 2-sulfamoylphenyl, 3-fluorophenyl, and        2-sulfamoyl-4-aminophenyl;    -   iii) —OR²⁰¹; for example, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂CH₃;    -   iv) —C(O)R²⁰¹; for example, —COCH₃, —COCH₂CH₃, —COCH₂CH₂CH₃;    -   v) —C(O)OR²⁰¹; for example, —CO₂CH₃, —CO₂CH₂CH₃, —CO₂CH₂CH₂CH₃;    -   vi) —C(O)N(R²⁰¹)₂; for example, —CONH₂, —CONHCH₃, —CON(CH₃)₂;    -   vii) —N(R²⁰¹)₂; for example, —NH₂, —NHCH₃, —N(CH₃)₂;        —NH(CH₂CH₃);    -   viii) halogen: —F, —Cl, —Br, and —I;    -   ix) —CH^(m′)X^(n′); wherein X is halogen, m′ is from 0 to 2,        m′+n′=3; for example, —CH₂F, —CHF₂, —CF₃, —CCl₃, or —CBr₃; and    -   x) —SO₂R²⁰¹; for example, —SO₂H; —SO₂CH₃; —SO₂C₆H₅; —SO₂NH₂;        wherein each R²⁰¹ is independently hydrogen, substituted or        unsubstituted C₁-C₄ linear, branched, or cyclic alkyl, amino,        alkylamino; or two R²⁰¹ units can be taken together to form a        ring comprising 3-7 atoms. Examples of this iteration include        compound having the formulae:

wherein R^(b) represents one or more optionally present substitutionsfor hydrogen as defined herein above.

The compounds wherein R^(5c) is a substituted or unsubstitutedcycloalkyl ring can have any enantiomeric or diastereomeric form. Forexample,

Another category relates to compounds wherein R^(5c) is a substituted orunsubstituted aryl ring. The following are non-limiting examples ofsubstituted aryl R^(5c) units:

2-Carboxyphenyl, 3-carboxyphenyl, 4-carboxyphenyl, 2,3-dicarboxyphenyl,2,4-dicarboxyphenyl, 2,5-dicarboxyphenyl, 2,6-dicarboxyphenyl,3,4-dicarboxyphenyl, 3,5-dicarboxyphenyl, 2,3,4-tricarboxyphenyl,2,3,5-tricarboxyphenyl, 2,3,6-tricarboxyphenyl, 2,4,6-tricarboxyphenyl,2,3,4,5-tetracarboxyphenyl, 2,3,4,6-tetracarboxyphenyl,2,3,4,5,6-pentacarboxyphenyl.

2-Fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 2,3-difluorophenyl,2,4-difluorophenyl, 2,5-difluorophenyl, 2,6-difluorophenyl,3,4-difluorophenyl, 3,5-difluorophenyl, 2,3,4-trifluorophenyl,2,3,5-trifluorophenyl, 2,3,6-trifluorophenyl, 2,4,6-trifluorophenyl,2,3,4,5-tetrafluorophenyl, 2,3,4,6-tetrafluorophenyl,2,3,4,5,6-pentafluorophenyl.

2-Chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 2,3-dichlorophenyl,2,4-dichlorophenyl, 2,5-dichlorophenyl, 2,6-dichlorophenyl,3,4-dichlorophenyl, 3,5-dichlorophenyl, 2,3,4-trichlorophenyl,2,3,5-trichlorophenyl, 2,3,6-trichlorophenyl, 2,4,6-trichlorophenyl,2,3,4,5-tetrachlorophenyl, 2,3,4,6-tetrachlorophenyl,2,3,4,5,6-pentachlorophenyl.

2-Bromophenyl, 3-bromophenyl, 4-bromophenyl, 2,3-dibromophenyl,2,4-dibromophenyl, 2,5-dibromophenyl, 2,6-dibromophenyl,3,4-dibromophenyl, 3,5-dibromophenyl, 2,3,4-tribromophenyl,2,3,5-tribromophenyl, 2,3,6-tribromophenyl, 2,4,6-tribromophenyl,2,3,4,5-tetrabromophenyl, 2,3,4,6-tetrabromophenyl,2,3,4,5,6-pentabromophenyl.

2-Iodophenyl, 3-iodophenyl, 4-iodophenyl, 2,3-diiodophenyl,2,4-diiodophenyl, 2,5-diiodophenyl, 2,6-diiodophenyl, 3,4-diiodophenyl,3,5-diiodophenyl, 2,3,4-triiodo-phenyl, 2,3,5-triiodophenyl,2,3,6-triiodophenyl, 2,4,6-triiodophenyl, 2,3,4,5-tetraiodo-phenyl,2,3,4,6-tetraiodophenyl, 2,3,4,5,6-pentaiodophenyl.

3-Hydroxyphenyl, 4-hydroxyphenyl, 2,3-dihydroxyphenyl,2,4-dihydroxyphenyl, 2,5-dihydroxyphenyl, 2,6-dihydroxyphenyl,3,4-dihydroxyphenyl, 3,5-dihydroxyphenyl, 2,3,4-trihydroxyphenyl,2,3,5-trihydroxyphenyl, 2,3,6-trihydroxy-phenyl, 2,4,6-trihydroxyphenyl,2,3,4,5-tetrahydroxyphenyl, 2,3,4,6-tetrahydroxyphenyl,2,3,4,5,6-pentahydroxyphenyl.

2-Methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2,3-dimethoxyphenyl,2,4-dimethoxyphenyl, 2,5-dimethoxyphenyl, 2,6-dimethoxyphenyl,3,4-dimethoxy-phenyl, 3,5-dimethoxyphenyl, 2,3,4-trimethoxyphenyl,2,3,5-trimethoxyphenyl, 2,3,6-trimethoxyphenyl, 2,4,6-trimethoxyphenyl,2,3,4,5-tetramethoxyphenyl, 2,3,4,6-tetra-methoxyphenyl,2,3,4,5,6-pentamethoxyphenyl.

2-Aminophenyl, 3-aminophenyl, 4-aminophenyl, 2,3-diaminophenyl,2,4-diaminophenyl, 2,5-diaminophenyl, 2,6-diaminophenyl,3,4-diaminophenyl, 3,5-diaminophenyl, 2,3,4-triaminophenyl,2,3,5-triaminophenyl, 2,3,6-triaminophenyl, 2,4,6-triaminophenyl,2,3,4,5-tetraaminophenyl, 2,3,4,6-tetraaminophenyl,2,3,4,5,6-pentaaminophenyl.

2-(Dimethylamino)phenyl, 3-(dimethylamino)phenyl,4-(dimethylamino)phenyl, 2,3-di(dimethylamino)phenyl,2,4-di(dimethylamino)phenyl, 2,5-di(dimethylamino)-phenyl,2,6-di(dimethylamino)phenyl, 3,4-di(dimethylamino)phenyl,3,5-di(dimethyl-amino)phenyl, 2,3,4-tri(dimethylamino)phenyl,2,3,5-tri(dimethylamino)phenyl, 2,3,6-tri(dimethylamino)phenyl,2,4,6-tri(dimethylamino)phenyl, 2,3,4,5-tetra(dimethylamino)-phenyl,2,3,4,6-tetra(dimethylamino)phenyl,2,3,4,5,6-penta(dimethylamino)phenyl.

One embodiment relates to compounds having the formula:

wherein R^(5c) is a substituted or unsubstituted aryl unit, for example,units having the formula:

wherein R^(c) and R^(d) each represents one or more optionally presentsubstitutions for hydrodgen. Non-limiting examples of R^(c) and R^(d)units include units independently chosen from:

-   -   i) C₁-C₄ linear or branched alkyl; for example, methyl (C₁),        ethyl (C₂), n-propyl (C₃), iso-propyl (C₃), n-butyl (C₄),        iso-butyl (C₄), sec-butyl (C₄), and tert-butyl (C₄);    -   ii) C₆ and C₁₀ substituted or unsubstituted aryl; for example,        4-aminophenyl, 2-sulfamoylphenyl, 3-fluorophenyl, and        2-sulfamoyl-4-aminophenyl;    -   iii) —OR²⁰²; for example, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂CH₃;    -   iv) —C(O)R²⁰²; for example, —COCH₃, —COCH₂CH₃, —COCH₂CH₂CH₃;    -   v) —C(O)OR²⁰²; for example, —CO₂CH₃, —CO₂CH₂CH₃, —CO₂CH₂CH₂CH₃;    -   vi) —C(O)N(R²⁰²)₂; for example, —CONH₂, —CONHCH₃, —CON(CH₃)₂;    -   vii) —N(R²⁰²)₂; for example, —NH₂, —NHCH₃, —N(CH₃)₂,        —NH(CH₂CH₃);    -   viii) halogen: —F, —Cl, —Br, and —I;    -   ix) —CH^(m′)X_(n′); wherein X is halogen, m′ is from 0 to 2,        m′+n′=3; for example, —CH₂F, —CHF₂, —CF₃, —CCl₃, or —CBr₃; and    -   x) —SO₂R²⁰²; for example, —SO₂H; —SO₂CH₃; —SO₂C₆H₅; —SO₂NH₂;        wherein each R²⁰² is independently hydrogen, substituted or        unsubstituted C₁-C₄ linear, branched, or cyclic alkyl, amino,        alkylamino; or two R²⁰² units can be taken together to form a        ring comprising 3-7 atoms.

Non-limiting examples of this embodiment includes2-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid having theformula:

3-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid having theformula:

4-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid having theformula:

2-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)benzamide having the formula:

8-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-y1)-1-naphthoic acid having theformula:

7-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-y1)-1-naphthoic acid having theformula:

and 2-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)-1-naphthoic acid havingthe formula:

Another embodiment relates to compounds having the formula:

wherein R^(5c) is substituted or unsubstituted aryl, R² and R^(2′) arethe same as defined herein above. Non-limiting examples include:

wherein R^(c) and R^(d) are the same as defined herein above.

A further embodiment of this category of compounds capable ofinteracting with at least one amino acid residue of the proteininteraction site of the β subunit of a G protein according to thepresent disclosure relates to compounds wherein R^(5a) and R^(5b) on acarbon atom are taken together to form an exocyclic double bond havingthe formula ═C(R^(5a′))(R^(5b′)), wherein R^(5a′) and R^(5b′) are eachindependently:

-   -   a) —H;    -   b) C₁-C₁₂ substituted or unsubstituted linear, branched, or        cyclic alkyl;    -   d) C₆ or C₁₀ substituted or unsubstituted aryl;    -   e) C₁-C₉ substituted or unsubstituted heterocyclic;    -   f) C₁-C₁₁ substituted or unsubstituted heteroaryl; or    -   g) R^(5a′) and R^(5b′) can be taken together to form a        substituted or unsubstituted ring having from 3 to 10 carbon        atoms and from 0 to 3 heteroatoms chosen from oxygen, nitrogen,        and sulfur.

A non-limiting example of a compound within this embodiment is(Z)-9-(3,3-dihydroxyallylidene)-4,5,6-trihydroxy-9,9a-dihydro-3H-xanthen-3-onehaving the formula:

Table 6 and FIG. 7 provide SIGK IC₅₀ data for non-limiting examples ofthis category of compounds capable of interacting with at least oneamino acid residue of the protein interaction site of the β subunit of aG protein according to the present disclosure.

TABLE 6 compound SIGK IC₅₀

 0.2 μM

 0.7 μM

  5 μM

 0.2 μM

 0.2 μM

0.15 μM

 0.2 μM

However during the course of the work leading to the present disclosure,we further investigated what modifications to the core ring systemhaving the formula:

would provide compounds capable of interacting with at least one aminoacid residue of the protein interaction site of the β subunit of a Gprotein. Disclosed herein above is the compound7-methyl-3-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)bicyclo[2.2.1]hept-5-ene-2-carboxylicacid having the formula:

which was found to bind with at least one amino acid residue of theprotein interaction site of the β subunit of a G protein at 700 nM. Weattached the R^(5c) unit 7-methyl-bicyclo[2.2.1]hept-5-ene-2-carboxylicacid to the 6-hydroxy-3H-xanthen-3-one ring system having the formula:

a ring system that was found not to provide compounds eliciting bindingwith certain R^(5c) units, to prepare3-(6-hydroxy-3-oxo-3H-xanthen-9yl)bicyclo[2.2.1]hept-5-ene-2-carboxylicacid having the formula:

This compound was found to bind at 14 μM.

Another category of compounds capable of interacting with at least oneamino acid residue of the protein interaction site of the β subunit of aG protein according to the present disclosure relates to compoundshaving the formula:

wherein X¹ is —[NR⁶]—, X⁴ is —[NH]—; X² and X³ are each —[CR^(14c)]═;and the R^(14c) unit from X² and X³ are taken together to form an arylring having 6 carbon atoms.

-   -   R¹, R², R³, R⁴, R^(1′), R^(2′), R^(3′) and R^(4′) are each        independently chosen from:    -   i) —H;    -   ii) C₆ or C₁₀ substituted or unsubstituted aryl;    -   iii) —OR²⁷;        -   R²⁷ is chosen from:        -   a) —H;        -   b) C₁-C₁₂ substituted or unsubstituted linear, branched, or            cyclic alkyl;    -   iv) —N(R^(28a))(R^(28b));        -   R^(28a) and R^(28b) are each independently chosen from:        -   a) —H;        -   d) C₆ or C₁₀ substituted or unsubstituted aryl;    -   v) halogen;    -   vi) —SO₂R³⁰;        -   R³⁰ is hydrogen, substituted or unsubstituted C₁-C₄ linear            or branched alkyl.

R⁶ is phenyl or substituted phenyl, the substitutions for hydrogen onthe phenyl ring are each independently chosen from:

-   -   i) C₁-C₄ linear or branched alkyl; for example, methyl (C₁),        ethyl (C₂), n-propyl (C₃), iso-propyl (C₃), n-butyl (C₄),        iso-butyl (C₄), sec-butyl (C₄), and tert-butyl (C₄);    -   ii) C₆ and C₁₀ substituted or unsubstituted aryl; for example,        4-aminophenyl, 2-sulfamoylphenyl, 3-fluorophenyl, and        2-sulfamoyl-4-aminophenyl;    -   iii) —OR¹⁰⁰; for example, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂CH₃;    -   iv) —C(O)R¹⁰⁰; for example, —COCH₃, —COCH₂CH₃, —COCH₂CH₂CH₃;    -   v) —C(O)OR¹⁰⁰; for example, —CO₂CH₃, —CO₂CH₂CH₃, —CO₂CH₂CH₂CH₃;    -   vi) —C(O)N(R¹⁰⁰)₂; for example, —CONH₂, —CONHCH₃, —CON(CH₃)₂;    -   vii) —N(R¹⁰⁰)₂; for example, —NH₂, —NHCH₃, —N(CH₃)₂,        —NH(CH₂CH₃);    -   viii) halogen: —F, —Cl, —Br, and —I;    -   ix) —CH^(m′)X^(n′); wherein X is halogen, m′ is from 0 to 2,        m′+n′=3; for example, —CH₂F, —CHF₂, —CF₃, —CCl₃, or —CBr₃; and    -   x) —SO₂R¹⁰⁰; for example, —SO₂H; —SO₂CH₃; —SO₂C₆H₅; —SO₂NH₂;        wherein each R¹⁰⁰ is independently hydrogen, substituted or        unsubstituted C₁-C₄ linear, branched, or cyclic alkyl, amino,        alkylamino; or two R¹⁰⁰ units can be taken together to form a        ring comprising 3-7 atoms. Substituents suitable for replacement        of a hydrogen atom are further defined herein below in the        iterations and examples.

Non-limiting examples of this embodiment include3,7-dimethyl-N²-phenyl-10-(4-methylphenyl)-5,10-dihydrophenazine-2,8-diaminehaving the formula:

10-phenyl-5,10-dihydrophenazine-2,8-diamine having the formula:

and N²,N²-dimethyl-10=phenyl-5,10-dihydrophenazine-2,8-diamine havingthe formula:

A further category of compounds capable of interacting with at least oneamino acid residue of the protein interaction site of the β subunit of aG protein according to the present disclosure relates to compoundshaving the formula:

wherein L is a linking group capable of linking a first ring system witha second ring system through any of the R¹, R², R³, R⁴, X¹, X², X³, orX⁴ units of each system, L having the formula:—[C(R^(23a))(R^(23b))]_(j)[W]_(k)[C(R^(24a))(R^(24b))]_(m)[Z]_(n)[C(R^(25a))(R^(25b))]_(o)—

-   R^(23a), R^(23b), R^(24a), R^(24b), R^(25a), and R^(25b) are each    independently chosen from;    -   i) —H; or    -   ii) C₁-C₄ substituted or unsubstituted linear or branched alkyl;    -   iii) C₇-C₁₅ substituted or unsubstituted arylalkylene;-   W and Z are each independently chosen from:    -   i) -M-;    -   ii) —C(=M)-;    -   iii) —C(=M)M-;    -   iv) -MC(=M)-;    -   v) -MC(=M)M-;    -   vi) -MC(=M)C(=M)M-;    -   vii) -MC(=M)MC(=M)M-;        each M is independently chosen from O, S, and NR²⁶; R²⁶ is        hydrogen, hydroxyl, or C₁-C₄ linear or branched alkyl. The        indices k and n are 0 or 1; the indices j, m, and o are from 0        to 12

One embodiment of compounds that comprise two linked ring systems hasthe formula:

wherein R¹, R², R⁴, X¹, X², X³, and X⁴ are the same as defined herein.

A first example of this embodiment relates to compounds wherein L hasthe formula:—[W][C(R^(24a))(R^(24b))]_(m)[Z]—wherein W and Z are each independently chosen from:

-   -   i) —NH—;    -   ii) —NHC(O)—; and    -   iii) —C(O)NH—.    -   R^(24a) and R^(24b) are each independently chosen from:    -   i) —H; or    -   ii) C₁-C₄ substituted or unsubstituted linear or branched alkyl;    -   iii) C₆ substituted or unsubstituted aryl; or    -   iv) C₇-C₁₅ substituted or unsubstituted arylalkylene;        the index m is from is from 0 to 6.

A first iteration of this embodiment relates to compounds wherein theindex m is equal to 0, W is —NH—, and Z is —C(O)NH—, the linking unit Lhaving the formula:—NHC(O)NH—.Non-limiting examples of compounds linked via a urea linking unitincludes compounds having the formula:

wherein each X¹ is CR^(5c) or N; R^(5c) chosen from hydrogen; C₁-C₄linear, branched, or cyclic alkyl; —N(R^(8a))(R^(8b)), R^(8a) and R^(8b)are each independently hydrogen or C₁-C₄ linear, branched, or cyclicalkyl; or R^(8a) and R^(8b) can be taken together to form a ring havingfrom 3 to 10 carbon atoms; R^(14c) is hydrogen or C₁-C₄ linear,branched, or cyclic alkyl.

Specific examples of this iteration includes 1,3-di(quinoline-6-yl)ureahaving the formula:

and 1,3-bis(4-amino-2-methylquinolin-6-yl)urea having the formula:

A further embodiment of this category relates to compounds having theformula:

wherein linking unit L has the formula:—[NHC(O)][C(R^(24a))(R^(24b))]_(m)[C(O)NH]—

-   R^(24a) and R^(24b) are each independently chosen from:    -   i) —H; or    -   ii) C₁-C₄ substituted or unsubstituted linear or branched alkyl.        the index m is from 1 to 6.

A first iteration of this embodiment relates to compounds having theformula:

wherein each R^(5c) and each R^(14c) is independently chosen from:

-   -   i) hydrogen;    -   ii) C₁-C₄ linear, branched, or cyclic alkyl;    -   iii) —N(R^(8a))(R^(8b)); R^(8a) and R^(8b) are each        independently hydrogen or C₁-C₄ linear, branched, or cyclic        alkyl; or R^(8a) and R^(8b) can be taken together to form a ring        having from 3 to 10 carbon atoms.

Non-limiting examples of this embodiment includesN¹,N³-bis(4-amino-2-methylquinolin-6-yl)-2,2-dimethylmalonamide havingthe formula:

N¹,N⁵-bis(4-amino-2-methylquinolin-6-yl)glutaramide having the formula:

and N¹,N⁷-bis(4-amino-2-methylquinolin-6-yl)heptandiamide having theformula:

Another embodiment of compounds that comprise two linked ring systemsare compounds wherein the linking group L is —NH—. A non-limitingexample of this embodiment includesbis(8-iodo-10-phenyl-5,10-dihydrophenazin-2-yl)amine having the formula:

Table 7 provides ELISA IC₅₀ data for non-limiting examples of thiscategory of compounds capable of interacting with at least one aminoacid residue of the protein interaction site of the β subunit of a Gprotein according to the present disclosure.

TABLE 7 compound ELISA IC₅₀

 μM

24 μM

74 μM

 μM

 μM

 2 μM

Other compounds which comprise the present disclosure include1,1′-(5,10-dihydroanthra[9,1,2-cde]benzo[rst]pentaphene-16,17-diyl)bis(azanediyl)dianthracne-9,10-dionehaving the formula:

Accordingly, one embodiment of the present invention is a compoundhaving a structure of Formula I:

Exemplary compounds having the structure of Formula I which depictvarious substituent R groups include, but are not limited to, thefollowing:

and pharmaceutically acceptable salts and complexes thereof.

Another embodiment of the present invention is a compound having astructure of Formula II:

Exemplary compounds having the structure of Formula II which depictvarious substituent R groups include, but are not limited to, thefollowing:

and pharmaceutically acceptable salts and complexes thereof.

Additional exemplary compounds which bind to the protein interactionsite of Gβ include, but are not limited to, the following:

and pharmaceutically acceptable salts and complexes thereof.

Exemplary compounds disclosed herein are intended to include allenantiomers, isomers or tautomers, as well as any derivatives of suchcompounds that retain the same biological activity as the originalcompound.

Exemplary compounds of the present invention were initially selectedfrom a computational screen to identify ligands that bind to the novelprotein interaction site of Gβ. The computational screen involved usingSYBYL molecular modeling software to model the protein interaction siteof Gβ as determined in the X-ray structure disclosed herein. Thecomputational docking screen was performed with the National CancerInstitute 1900 compound library wherein the compounds were tested fordocking to the protein interaction site of Gβ using FLEXX™ (Tripos,Inc., St. Louis, Mo.) for docking and CSCORE™ (Tripos, Inc.) to evaluatethe energetics and fitness of the docked structure. Algorithm-dependentlists of compounds, predicted to interact with the protein interactionsite of Gβ and the structural model of the interaction, were generated.Selected compounds were subsequently analyzed in the phage ELISA bindingassay disclosed herein to assess whether these compounds could bind tothe protein interaction site of Gβ and interfere with proteininteractions at this surface. Compounds NSC201400 and NSC119916 had IC₅₀values of.100 nM and 5 μM, respectively, and the remaining compoundswere found to bind in the ELISA-based assay to Gβγ with an affinity ofat least 50 μM and interfere with peptide interactions at the proteininteraction site (FIG. 2). These compounds were further analyzed in thephage ELISA assay and found to have high affinities for the proteininteraction site of Gβ and interacted with similar amino acid residuesas SIGK.

TABLE 8 SIGK NSC30820 NSC12155 NSC117079 NSC23128 NSC402959 NSC109268Lys57 Lys57 Lys57 Lys57 Tyr59 Tyr59 Tyr59 Tyr59 Gln75 Gln75 Trp99 Trp99Trp99 Trp99 Val100 Met101 Leu117 Leu117 Tyr145 Asp186 Met188 Cys204Asp228 Asp228 Asn230 Asn230 Asn230 Asn230 Asp246 Asp246 Asp246 Thr274Arg314 Arg314 Trp332 IC₅₀ 100 nM 13 μM 43 μM 16 μM 2 μM 13 μM SIGKNSC125910 NSC119910 NSC30671 Lys57 Tyr59 Lys57 Lys57 Tyr59 Tyr59 Tyr59Trp99 Trp99 Val100 Trp99 Trp99 Met101 Val100 Leu117 Tyr145 Leu117 Met101Asp186 Leu117 Met188 Met188 Asp228 Asn230 Cys204 Asp246 Asp228 Thr274Trp332 Trp332 Ser316 Trp332 IC₅₀ 68 μM 100 nM 7 μM Underlined residuesindicate residues important for the SIGK*Gβ1γ₂ interaction. The last rowindicates the IC₅₀ value for each compound.

To further illustrate the utility of these compounds, it wasdemonstrated that NSC119910 blocked interactions of Ga subunit with Gβγsubunits (FIG. 3) and inhibited the ability of Gβγ subunits to inhibitinteractions with a physiological target such as PLC β in vitro (FIG. 4)based on a decrease in the enzymatic activity of PLC β. Gβγ-regulatedactivities of PI3Kγ and PLC-β2/-β3 are important inchemoattractant-induced responses and inflammation. PI3Kγ is involved inthe production of TI-Igλ_(L) and mice deficient in PI3Kγ, lackneutrophil migration (Li, et al. (2000) Science 287:1046-9). The PLCpathway is involved in down-modulation of chemotaxis and inhyperinflammatory conditions (Li, et al. (2000) supra). Therefore, itwas determined whether NSC119910 could inhibit the Gβy/PLC interactionand block PLC activation. Data from fura-2-based experimentsdemonstrated that the abruptly occurring increase in cytosolic Ca²⁺infMLP-stimulated neutrophils, a response which is due to the release ofthe cation from intracellular stores (Anderson, and Mahomed (1997) Clin.Exp. Immunol. 110:132-138; Geiszt, et al. (1997) J. Biol. Chem.272:26471-26478), was suppressed by 10 μM NSC119910. Increases in [Ca²⁺]through ATP was not significantly suppressed in the presence ofNSC119910, indicating that the effect of the compound on fMLP dependentCa²⁺ increases are specific. Further, the time taken for fluorescence todecline to half-peak values was not substantially affected. The resultsindicate that NSC 119910 inhibits PLC/G-protein interactions which leadto activation of PLC in vivo.

Opioid receptors, μ, Δ, and K, couple to Gi and G₀ proteins through αand βy subunits, and regulate a number of signaling pathways. Inparticular, the efficacy of opioid signal transduction inPLC-β3-deficient mice has been shown to increase, indicating that PLCsuppresses opioid signaling by modification of opioid-dependentsignaling components (Xie, et al. (1999) Proc. Natl. Acad. Sci. USA96:10385-10390). Given that PLC-β3 plays a significant role as anegative regulator of opioid responses, it was determined whetherNSC119910 could inhibit PLC-β3 activation thereby enhancingmorphine-induced analgesia. Mice were intracerebroventricularly injectedin accordance with standard protocols (Xu, et al. (1998) J. Pharmacol.Exp. Therapeut. 284:196-201) with 100 nmol of NSC119910 in combinationwith varying doses (0.1, 0.3, 1, and 3 nmol) of morphine. Mice weretested 20 minutes after the injection for an analgesic response in a 55°C. warm-water tail-flick test (Wells, et al. (2001) J. Pharmacol. Exp.Therapeut. 297:597-605). The ED₅₀ value for morphine alone was 0.74nmol, while the ED₅₀ value for NSC119910 plus morphine was 0.065 nmol.The differences in the ED₅₀ values showed an 11-fold shift to the leftin a morphine dose-response curve (Table 9), indicating that whenmorphine was administered with NSC119910, less morphine was required toproduce a similar analgesic effect. Accordingly, administering opioidsin combination with a compound of the instant invention would allow forthe use of a lower dose of opioid in patients thereby reducing thedevelopment of opioid tolerance.

TABLE 9 Percent Antinociception ± S.E.M. Dose of Morphine, nmol MorphineAlone Morphine plus NSC119910 10 82.4 ± 11.9 N/A 3 68.0 ± 14.1 100 ±0.0  1 55.6 ± 8.3  79.3 ± 9.1  0.3 41.0 ± 10.2 64.4 ± 10.0 0.1 21.0 ±11.1 55.3 ± 12.7

Having demonstrated that NSC119910 effectively modulates G-proteininteractions, a series of structural analogs of NSC119910, identifiedusing modeling software, were analyzed for binding to the proteininteraction site of Gβ. These analogs and their corresponding affinitiesfor Gβγ were:

From this analysis, a general structure for NSC119910 analogs wasidentified and is represented as Formula III.

wherein, Ri can be a substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted cycloalkyl, orsubstituted or unsubstituted cycloalkenyl; and R₂ and R₃ areindependently hydrogen or a hydroxyl group. In particular embodiments,R₂ and R₃ are both hydroxyl.

As used herein, alkyl refers to a straight or branched hydrocarbon chainconsisting solely of carbon and hydrogen atoms, containing nosaturation, having from one to eight carbon atoms.

Alkenyl is intended to mean an aliphatic hydrocarbon group containing atleast one carbon-carbon double bond and which may be a straight orbranched chain having from 2 to about 10 carbon atoms.

Cycloalkyl denotes a non-aromatic mono or multicyclic ring system ofabout 3 to 12 carbon atoms.

As used herein, the term cycloalkenyl refers to a mono or multicyclicring system containing in the range of about 3 to 12 carbon atoms withat least one carbon-carbon double bond.

Substituents in the substituted alkyl, cycloalkyl, alkenyl orcycloalkenyl groups include, but are not limited to, hydroxy, carboxyl,halogen (e.g., fluorine, chlorine, bromine, or iodine), or substitutedor unsubstituted alkyl. With the exception of NSC 157411 and NSC 122390,analogs of NSC119910 generally contained hydroxyl groups in the R₂ andR₃ positions of Formula III, which appeared to facilitate binding; and acarboxyl-substituted alkyl, cycloalkyl, alkenyl or cycloalkenylsubstituent at R₁ of Formula III, which appeared to modulate activity,but was not required for binding.

Accordingly, a further embodiment of the present invention is a compoundhaving a structure of Formula III and pharmaceutically acceptable saltsand complexes thereof.

C. Methods of Using the Compositions

1. Methods of Treating Disease

Chemoattractant-mediated recruitment of leukocytes is responsible formany of the deleterious effects of chronic inflammatory diseases. Manychemoattractants activate G protein-coupled receptors (GPCRs) coupled tothe Gi family of heterotrimeric G proteins in leukocytes. HeterotrimericG proteins are composed of Gα, Gβ, and Gγ subunits. Ligand binding toreceptors catalyzes the exchange of tightly bound GDP for GTP on the Gαsubunit, liberating it from the Gβγ subunits. Dissociation of the Gα andGβγ subunits can allow each to directly bind to downstream effectorproteins (Gilman, 1987; Oldham and Hamm, 2006). The free Gβγ subunitsreleased from Gi heterotrimers upon chemoattractant receptor activationinitiate critical signaling pathways to direct chemoattractant-dependentneutrophil functions including chemotaxis and superoxide production(Neptune and Bourne, 1997).

Key direct targets of Gβγ subunit binding and activation in neutrophilsare phosphoinositide 3-kinase γ(PI3-kinase γ) (Stephens et al., 1994,1997; Stoyanov et al., 1995), Phospholipase C β (PLCβ) (Wu et al.,2000), and P-Rex (Welch et al., 2002). PI3-kinase γ has been noted to bea central mediator of chemotaxis and plays a pivotal role in leukocyterecruitment to inflamed tissues (Hirsch et al., 2000; Li et al., 2000;Camps et al., 2005). PIP3, produced by PI3-kinase γ catalytic activity,is critical to the development of cell polarity, which is necessary forchemokine-mediated cell motility and directional sensing (Wu et al.,2000). PI3-kinase γ-deficient neutrophils have impaired responses tovarious chemoattractants, including diminished chemotaxis (Hirsch etal., 2000; Li et al., 2000) and respiratory burst (Li et al., 2000;Sasaki et al., 2000), in response to GPCR activation. Small-moleculeinhibitors of PI3-kinase γ catalytic activity have been demonstrated tosuppress joint inflammation in mouse models of inflammation (Barber etal., 2005; Camps et al., 2005). The development of selective inhibitorsthat do not target other PI3-kinase isoforms is important to the successof a method that targets PI3-kinase γ activity as a therapeuticanti-inflammatory approach, because these enzymes are involved inmultiple aspects of mammalian cell function (Rückle et al., 2006).

Herein disclosed are novel strategies to inhibitchemoattractant-dependent chemotaxis and inflammation using recentlyidentified compounds that block Gβγ-interactions with effectors,including PI3-kinase γ, by binding to a protein-protein interaction “hotspot” on the Gβ subunit (Bonacci et al., 2006). These compounds blockfMLP-dependent PI3-kinase γ activation, Rac1 activation, superoxideproduction, and neutrophil migration in vitro. Furthermore, whenadministered in vivo, neutrophil-dependent inflammation is inhibited,demonstrating that suppressing key Gβγ-dependent signaling functionswith small molecules has significant anti-inflammatory potential.

The ability of NSC119910 analogs to selectively modulate activationPLC-β2 and -β3 was analyzed. In this assay, PLC-β2 and PLC-β3 werepurified and PLC enzymatic activity was measured in the presence orabsence of purified βy and in the presence or absence of analog. Theresults of this analysis indicated that NSC119911 appeared to blockPLC-β2 activation more effectively than PLC-β3 activation and NSC201400selectively potentiated PLC-β3 activation despite blocking peptidebinding to βγ (FIG. 6). Further, while NSC119910, NSC and analogNSC119893 block Ca²⁺ mobilization, they do so without interfering withfMLP-dependent ERK activation. Likewise, NSC119911, NSC158110, andNSC201400 also do not interfere with fMLP-dependent ERK activation.

The compounds disclosed herein as well as those found to bind to theprotein interaction site of Gβ and interfere with protein interactionsat this surface can be used in a method for modulating (i.e., blockingor inhibiting, or enhancing or potentiating) at least one activity of aG protein. Such a method involves contacting a G protein either in vitroor in vivo with an effective amount of an agent that interacts with atleast one amino acid residue of the protein interaction site of the Gprotein β subunit so that at least one activity of the G protein ismodulated. An effective amount of an agent is an amount which reduces orincreases the activity of the G protein by 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or 100%. Such activity can be monitored based onprotein-protein interactions or enzymatic assays detecting activity ofdownstream proteins.

As will be appreciated by one of skill in the art, modulating one ormore G protein activities can be useful in selectively analyzing Gprotein signaling events in model systems as well as in preventing ortreating diseases and disorders involving G protein βy subunitsignaling. The selection of the compound for use in preventing ortreating a particular disease or disorder will be dependent upon theparticular G protein-dependent downstream protein involved in thedisease or disorder. For example, a compound which interacts with Lys57,Trp99, Met101, Leu117, Asp186, Asp228, Asp246 and/or Trp332 of Gβ wouldbe useful in preventing or treating adenylyl cyclase-associated diseasesor disorders, whereas a compound which interacts with Lys57, Tyr59,Trp99, Met101, Leu117, Tyr146, Met188, Asp246, and/or Trp332 may be moresuitable for GRK2-associated diseases or disorders. It is contemplatedthat this selectivity for specific downstream proteins may reduce sideeffects associated with antagonists which inhibit all activitiesassociated the G protein βγ subunit signaling.

Prevention or treatment typically involves the steps of firstidentifying a patient at risk of having or having a disease or disorderinvolving at least one G protein βy subunit activity {e.g., congestiveheart failure, addiction, hyper- or hypo-inflammation, or opioidtolerance). Once such an individual is identified using, for example,standard clinical practices, said individual is administered apharmaceutical composition containing an effective of a selectivecompound disclosed herein or identified in the screening methods of theinvention. In most cases this will be a human being, but treatment ofagricultural animals, e.g., livestock and poultry, and companionanimals, e.g., dogs, cats and horses, is expressly covered herein. Theselection of the dosage or effective amount of a compound is that whichhas the desired outcome of reducing or reversing at least one sign orsymptom of a disease or disorder involving G protein βy subunitsignaling in a patient. For example, some of the general signs orsymptoms associated with congestive heart failure include increasedheart rate, increased respiratory rate, breathing faster and deeper thannormal, breathlessness, irritability, restlessness, an unexplainedfussiness, swelling, puffiness, edema, sudden weight gain or poor weightgain, decrease in appetite, diaphoresis, cough, congestion or wheezing,a decrease in activity level, fatigue, listlessness, decrease in urineoutput, or pale, mottled or grayish appearance in skin color. Generalsigns or symptoms associated with addiction include, but are not limitedto, changes in attitude, appearance, and relationships with others,whether at home, school or work and other behavioral changes.

When preventing or treating an inflammatory condition, the selectivemodulation of either the PLC pathway or PI3Kγ will be useful in treatingdifferent inflammatory conditions. For example, to reduce neutrophilmigration into sites of inflammation (e.g., in arthritis) it isdesirable to administer a compound which selectively inhibits theactivation of PI3Kγ thereby reducing the injury to tissues thatcontribute to the pathophysiology of the inflammatory diseases.Conversely, to facilitate an inflammatory response, e.g., to enhanceimmune responses to bacterial or viral infection, it is desirable toadminister a compound which selectively inhibits the activation of thePLC pathway.

By “subject” is meant an individual. Preferably, the subject is a mammalsuch as a primate, and, more preferably, a human. The term “subject” caninclude domesticated animals, such as cats, dogs, etc., livestock (e.g.,cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g.,mouse, rabbit, rat, guinea pig, etc.).

The present methods can also be treat an autoimmune or inflammatorycondition. Such conditions include but are not limited to asthma,rheumatoid arthritis, reactive arthritis, spondylarthritis, systemicvasculitis, insulin dependent diabetes mellitus, multiple sclerosis,experimental allergic encephalomyelitis, Sjögren's syndrome, graftversus host disease, inflammatory bowel disease including Crohn'sdisease, ulcerative colitis, ischemia reperfusion injury, Alzheimer'sdisease, transplant rejection (allogeneic and xenogeneic), thermaltrauma, any immune complex-induced inflammation, glomerulonephritis,myasthenia gravis, cerebral lupus, Guillaine-Barre syndrome, vasculitis,systemic sclerosis, anaphylaxis, catheter reactions, atheroma,infertility, thyroiditis, ARDS, post-bypass syndrome, hemodialysis,juvenile rheumatoid, Behcets syndrome, hemolytic anemia, pemphigus,bulbous pemphigoid, stroke, atherosclerosis, and scleroderma.

It is understood and herein contemplated that seizures can be treatedusing the methods and compounds disclosed herein. There are a numerouscauses for seizures known in the art. For example, seizures can be theresult of inflammation. For example, the seizures can be associated withhippocampal sclerosis, tuberous sclerosis, or cortical dysplasia.Additionally, seizures are associated with infections diseases such asNeisseria meningitides, Listeria monocytogenes, Streptococcuspneumoniae, Lymphocytic choriomeningitis virus, Herpes Simplex virustype-1, West Nile virus, Ebola virus, Marburg virus, Sin Nombre virus,Rift Valley Fever, Hantavirus, Blackcreek canal virus, Lassa virus,Junin virus, Machupo virus, Mumps, Measles, and Influenza. Thusdisclosed herein are methods of treating seizures in a subject in needthereof comprising administering to the subject an agent that interactswith at least one amino acid residue of the protein interaction site ofthe G protein β subunit, and wherein the agent modulates one or more Gprotein β subunit activities. Also disclosed are methods wherein theseizures are associated with hippocampal sclerosis, tuberous sclerosis,or cortical dysplasia. Also disclosed are methods wherein the seizuresare associated with an infectious disease, the infectious disease isselected from the group consisting of Neisseria meningitides, Listeriamonocytogenes, Streptococcus pneumoniae, Lymphocytic choriomeningitisvirus, Herpes Simplex virus type-1, West Nile virus, Ebola virus,Marburg virus, Sin Nombre virus, Rift Valley Fever, Hantavirus,Blackcreek canal virus, Lassa virus, Junin virus, Machupo virus, Mumps,Measles, and Influenza.

The disclosed methods utilize tissue samples from the subject to providethe basis for assessment. Such tissue samples can include, but are notlimited to, blood (including peripheral blood and peripheral bloodmononuclear cells), tissue biopsy samples (e.g., spleen, liver, bonemarrow, thymus, lung, kidney, brain, salivary glands, skin, lymph nodes,and intestinal tract), and specimens acquired by pulmonary lavage (e.g.,bronchoalveolar lavage (BAL)). Thus it is understood that the tissuesample can be from both lymphoid and non-lymphoid tissue. Examples ofnon-lymphoid tissue include but are not limited to lung, liver, kidney,and gut. Lymphoid tissue includes both primary and secondary lymphoidorgans such as the spleen, bone marrow, thymus, and lymph nodes.

Heart failure (HF) is the leading cause of death worldwide, the leadingcause of hospitalization in the US, and is predicted to be the leadingcause of disability by 2020. Five-year survival after HF diagnosis is50%, and 1-year survival for end-stage HF is only 50%.

Despite therapeutic advances of the past two decades, β-adrenergicreceptors (β-AR) play a central role in cardiac contractility, and aredramatically down-regulated and desensitized in end-stage HF. Elevatedexpression and activity of the G-protein coupled receptor kinase 2(GRK2, or βARK1), a molecule responsible for chronic β-ARdesensitization, is a hallmark of HF and can directly cause HF in animalmodels. Moreover GRK2 expression and activity correlates with theseverity of human HF.

GRK2 is a cytosolic enzyme recruited to membrane-bound Gβγ followingβ-AR stimulation; the Gβγ-GRK2 interaction is a prerequisite forGRK2-mediated β-AR desensitization. Specific Gβγ-GRK2 inhibitorycompounds can be identified by molecular modeling, and a compound namedM119 was found that blocks in vitro Gβγ-GRK2 interaction (Bonacci et al,Science, 2006). Large peptide inhibitors of Gβγ and the Gβγ-GRK2interaction, including the GRK2 C-terminus (βARKct) or truncatedphosducin can prevent and reverse HF in animal models by viral genedelivery. β-blockers, standard therapy in medical HF management, aresynergistic with βARKct. Thus, M119 inhibition of the Gβγ-GRK2interaction is a promising therapeutic strategy for HF, and offersdistinct size, delivery and specificity advantages over viral genedelivery of large peptides as HF therapy.

The cardiac effects of M119 were investigated in vitro and in vivo.Using adult mouse cardiomyocytes, it was found that M119 reduces β-ARstimulated membrane recruitment of GRK2, and enhances cAMP generation atbaseline and in response to β-AR stimulation. Increased cardiomyocytecontractility was demonstrated at baseline and in response to β-ARagonist. Importantly, normalization of cardiac function, morphology andGRK2 expression is demonstrated in an acute animal model of HF.

PI3Kγ, the only PI3K regulated by Gβγ, is involved in β-ARdesensitization by GRK2-PI3Kγ complex recruitment to Gβγ. Large peptidedisruption of PI3Kγ and its interaction with GRK2 is cardioprotective:genetic ablation of PI3Kγ is not. Compounds were identified thatdifferentially affect Gβγ-GRK2, Gβγ-PI3K, or both. These new chemicaltools are used to test whether PI3Kγ regulation of β-AR signaling isGβγ- and/or GRK2 dependent or independent.

Thus, it is contemplated herein that present methods can also be used totreat conditions associated with heart malfunction. In particular, it iscontemplated herein that the heart condition can be a cardiovascularindication associated with heart failure including but not limited tomyocardial infarction, restenosis, hypertension, and all primary andsecondary cardiomypathies, including but not limited to: dilated,(ischemic, non-ischemic, idiopathic, congestive, diabetic, peripartium,alcoholic, viral, and valvular) hypertrophic, and restrictive. Thus, forexample, disclosed herein are methods for treating a disease orcondition involving at least one G protein βγ subunit activitycomprising administering to a patient having or at risk of having adisease or condition involving at least one G protein βγ subunitactivity an effective amount of an agent that interacts with at leastone amino acid residue of the protein interaction site of the G proteinβ subunit so that the at least one activity of the G protein ismodulated thereby preventing or treating the disease or conditioninvolving the at least one G protein βy subunit activity, wherein thedisease or condition is a associated with heart malfunction and whereinthe disease of condition is selected from the group consisting ofmyocardial infarction, restenosis, hypertension, and all primary andsecondary cardiomypathies, including but not limited to: dilated,(ischemic, non-ischemic, idiopathic, congestive, diabetic, peripartium,alcoholic, viral, and valvular) hypertrophic, and restrictive.

Furthermore, it is understood and herein contemplated that diseasesaffecting the vasculature may affect heart function. Examples of suchvaculature diseases include but are not limited to: hypertension of anyetiology, atherosclerosis, peripheral vascular disease, restenosis.Thus, disclosed herein are methods for treating a disease or conditioninvolving at least one G protein βγ subunit activity comprisingadministering to a patient having or at risk of having a disease orcondition involving at least one G protein βγ subunit activity aneffective amount of an agent that interacts with at least one amino acidresidue of the protein interaction site of the G protein β subunit sothat the at least one activity of the G protein is modulated therebypreventing or treating the disease or condition involving the at leastone G protein βy subunit activity, wherein the disease or condition is adisease affecting vasculature, wherein in the disease is selected fromthe vasculature diseases consisting of hypertension of any etiology,atherosclerosis, peripheral vascular disease, and restenosis.

Due to the ability of the present methods to stabilize cardiac function,it is further contemplated herein that the methods and disclosedcompositions can be used as an adjunct to therapy or transplantation forany heart dysfunction, including but not limited to: ventricular assistdevice, implantable defibrillator, ventricular pacemakers,transplantation. Thus it is contemplated herein that the disclosedmethods and compositions can be used in conjunction with any methodknown in the art to treat heart dysfunction. For example, disclosedherein are methods of treating heart dysfunction in a subject comprisingadministering to the subject an effective amount of an agent thatinteracts with at least one amino acid residue of the proteininteraction site of the G protein β subunit so that the at least oneactivity of the G protein is modulated and administering to the subjecta therapy of transplantation to the subject such as a ventricular assistdevice, implantable defibrillator, or ventricular pacemaker.

2. Methods Relating to M-Opioid Analgesia and Antinociception

It is understood and herein contemplated that one use for compoundsidentified by the disclosed methods as well as the compounds and agentsdisclosed herein relates to the effect of G protein b subunit onμ-opioids such as morphine. In one aspect it is understood that thedisclosed compounds can enhance the analgesic effect of μ-opioids. Thus,disclosed herein are methods of screening for an agent that enhancesμ-opioid analgesia comprising contacting a G protein β subunit with atest agent and determining whether the agent interacts with at least oneamino acid residue of the protein interaction site of the β subunitthereby identifying an agent that enhances μ-opioid analgesia. Alsodisclosed are methods further comprising measuring the downstreamsignaling activity of PLCβ3, wherein a decreased activity indicates anagent that enhances μ-opioid analgesia. It is understood that inaddition to the ability to relieve pain through enhancing the analgesicproperties of μ-opioids, the disclosed compounds and methods candiminish the perception of pain when used in conjunction with μ-opioids.Thus, disclosed herein are method of modulating acute μ-opioid-dependentantinociception in a subject comprising administering to the subject aμ-opioid and an agent that interacts with at least one amino acidresidue of the protein interaction site of the G protein β subunit, andwherein the agent modulates one or more G protein β subunit activities.Also disclosed are methods wherein the agent increases theantinociception effects of the μ-opioid. Also disclosed are methodswherein the G protein β subunit activity is the signaling to PLCβ3, andwherein an inhibition of PLCβ3 activity indicates an agent that an agentthat increases antinociception.

Enhancing the analgesic and/or antinocicpetive effect of μ-opioids hasthe benefit of decreasing the amount of a μ-opioid needed to relievepain. Thus, additional benefits resulting from the disclosed methods andagents when used in conjunction with m-opioids is decreasing toleranceto the μ-opioid and decreasing the risk of dependence (i.e., addiction)on the μ-opioid. Thus disclosed herein are methods of decreasing therisk of dependence on μ-opioids in a subject in need thereof comprisingadministering to the subject a μ-opioid and an agent that interacts withat least one amino acid residue of the protein interaction site of the Gprotein β subunit, and wherein the agent modulates one or more G proteinβ subunit activities. Also disclosed are methods of decreasing toleranceto μ-opioids in a subject in need thereof comprising administering tothe subject a μ-opioid and an agent that interacts with at least oneamino acid residue of the protein interaction site of the G protein βsubunit, and wherein the agent modulates one or more G protein β subunitactivities. It is understood and herein contemplated that such agentscan also be used as anti-addiction medication.

3. Method of Treating Cancers

The disclosed compositions can be used to treat any disease whereuncontrolled cellular proliferation occurs such as cancers. Anon-limiting list of different types of cancers is as follows: lymphomas(Hodgkins and non-Hodgkins), leukemias, carcinomas, carcinomas of solidtissues, squamous cell carcinomas, adenocarcinomas, sarcomas, gliomas,high grade gliomas, blastomas, neuroblastomas, plasmacytomas,histiocytomas, melanomas, adenomas, hypoxic tumours, myelomas,AIDS-related lymphomas or sarcomas, metastatic cancers, or cancers ingeneral.

A representative but non-limiting list of cancers that the disclosedcompositions can be used to treat is the following: lymphoma, B celllymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloidleukemia, bladder cancer, brain cancer, nervous system cancer, head andneck cancer, squamous cell carcinoma of head and neck, kidney cancer,lung cancers such as small cell lung cancer and non-small cell lungcancer, neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer,prostate cancer, skin cancer, liver cancer, melanoma, squamous cellcarcinomas of the mouth, throat, larynx, and lung, colon cancer,cervical cancer, cervical carcinoma, breast cancer, and epithelialcancer, renal cancer, genitourinary cancer, pulmonary cancer, esophagealcarcinoma, head and neck carcinoma, large bowel cancer, hematopoieticcancers; testicular cancer; colon and rectal cancers, prostatic cancer,or pancreatic cancer.

Compounds disclosed herein may also be used for the treatment ofprecancer conditions such as cervical and anal dysplasias, otherdysplasias, severe dysplasias, hyperplasias, atypical hyperplasias, andneoplasias.

D. Homology/Identity

It is understood that one way to define any known variants andderivatives or those that might arise, of the disclosed genes andproteins herein is through defining the variants and derivatives interms of homology to specific known sequences. For example SEQ ID NO: 1sets forth a particular sequence of an SIRK peptide. Specificallydisclosed are variants of these and other genes and proteins hereindisclosed which have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99 percent homology to the stated sequence. Those of skill in theart readily understand how to determine the homology of two proteins ornucleic acids, such as genes. For example, the homology can becalculated after aligning the two sequences so that the homology is atits highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment.

E. Delivery of the Compositions to Cells

There are a number of compositions and methods which can be used todeliver nucleic acids to cells, either in vitro or in vivo. Thesemethods and compositions can largely be broken down into two classes:viral based delivery systems and non-viral based delivery systems. Forexample, the nucleic acids can be delivered through a number of directdelivery systems such as, electroporation, lipofection, calciumphosphate precipitation, plasmids, viral vectors, viral nucleic acids,phage nucleic acids, phages, cosmids, or via transfer of geneticmaterial in cells or carriers such as cationic liposomes. Appropriatemeans for transfection, including viral vectors, chemical transfectants,or physico-mechanical methods such as electroporation and directdiffusion of DNA, are described by, for example, Wolff, J. A., et al.,Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818,(1991). Such methods are well known in the art and readily adaptable foruse with the compositions and methods described herein. In certaincases, the methods will be modified to specifically function with largeDNA molecules. Further, these methods can be used to target certaindiseases and cell populations by using the targeting characteristics ofthe carrier.

1. Nucleic Acid Based Delivery Systems

Transfer vectors can be any nucleotide construction used to delivergenes into cells (e.g., a plasmid), or as part of a general strategy todeliver genes, e.g., as part of recombinant retrovirus or adenovirus(Ram et al. Cancer Res. 53:83-88, (1993)).

As used herein, plasmid or viral vectors are agents that transport thedisclosed nucleic acids, such as SIRK, SIGK, and βARKct into the cellwithout degradation and include a promoter yielding expression of thegene in the cells into which it is delivered. In some embodiments theSIRK, SIGK, and βARKct s are derived from either a virus or aretrovirus. Viral vectors are, for example, Adenovirus, Adeno-associatedvirus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus, neuronaltrophic virus, Sindbis and other RNA viruses, including these viruseswith the HIV backbone. Also preferred are any viral families which sharethe properties of these viruses which make them suitable for use asvectors. Retroviruses include Murine Maloney Leukemia virus, MMLV, andretroviruses that express the desirable properties of MMLV as a vector.Retroviral vectors are able to carry a larger genetic payload, i.e., atransgene or marker gene, than other viral vectors, and for this reasonare a commonly used vector. However, they are not as useful innon-proliferating cells. Adenovirus vectors are relatively stable andeasy to work with, have high titers, and can be delivered in aerosolformulation, and can transfect non-dividing cells. Pox viral vectors arelarge and have several sites for inserting genes, they are thermostableand can be stored at room temperature. A preferred embodiment is a viralvector which has been engineered so as to suppress the immune responseof the host organism, elicited by the viral antigens. Preferred vectorsof this type will carry coding regions for Interleukin 8 or 10.

Viral vectors can have higher transaction (ability to introduce genes)abilities than chemical or physical methods to introduce genes intocells. Typically, viral vectors contain, nonstructural early genes,structural late genes, an RNA polymerase III transcript, invertedterminal repeats necessary for replication and encapsidation, andpromoters to control the transcription and replication of the viralgenome. When engineered as vectors, viruses typically have one or moreof the early genes removed and a gene or gene/promotor cassette isinserted into the viral genome in place of the removed viral DNA.Constructs of this type can carry up to about 8 kb of foreign geneticmaterial. The necessary functions of the removed early genes aretypically supplied by cell lines which have been engineered to expressthe gene products of the early genes in trans.

a) Retroviral Vectors

A retrovirus is an animal virus belonging to the virus family ofRetroviridae, including any types, subfamilies, genus, or tropisms.Retroviral vectors, in general, are described by Verma, I. M.,Retroviral vectors for gene transfer. In Microbiology-1985, AmericanSociety for Microbiology, pp. 229-232, Washington, (1985), which isincorporated by reference herein. Examples of methods for usingretroviral vectors for gene therapy are described in U.S. Pat. Nos.4,868,116 and 4,980,286; PCT applications WO 90/02806 and WO 89/07136;and Mulligan, (Science 260:926-932 (1993)); the teachings of which areincorporated herein by reference.

A retrovirus is essentially a package which has packed into it nucleicacid cargo. The nucleic acid cargo carries with it a packaging signal,which ensures that the replicated daughter molecules will be efficientlypackaged within the package coat. In addition to the package signal,there are a number of molecules which are needed in cis, for thereplication, and packaging of the replicated virus. Typically aretroviral genome, contains the gag, pol, and env genes which areinvolved in the making of the protein coat. It is the gag, pol, and envgenes which are typically replaced by the foreign DNA that it is to betransferred to the target cell. Retrovirus vectors typically contain apackaging signal for incorporation into the package coat, a sequencewhich signals the start of the gag transcription unit, elementsnecessary for reverse transcription, including a primer binding site tobind the tRNA primer of reverse transcription, terminal repeat sequencesthat guide the switch of RNA strands during DNA synthesis, a purine richsequence 5′ to the 3′ LTR that serve as the priming site for thesynthesis of the second strand of DNA synthesis, and specific sequencesnear the ends of the LTRs that enable the insertion of the DNA state ofthe retrovirus to insert into the host genome. The removal of the gag,pol, and env genes allows for about 8 kb of foreign sequence to beinserted into the viral genome, become reverse transcribed, and uponreplication be packaged into a new retroviral particle. This amount ofnucleic acid is sufficient for the delivery of a one to many genesdepending on the size of each transcript. It is preferable to includeeither positive or negative selectable markers along with other genes inthe insert.

Since the replication machinery and packaging proteins in mostretroviral vectors have been removed (gag, pol, and env), the vectorsare typically generated by placing them into a packaging cell line. Apackaging cell line is a cell line which has been transfected ortransformed with a retrovirus that contains the replication andpackaging machinery, but lacks any packaging signal. When the vectorcarrying the DNA of choice is transfected into these cell lines, thevector containing the gene of interest is replicated and packaged intonew retroviral particles, by the machinery provided in cis by the helpercell. The genomes for the machinery are not packaged because they lackthe necessary signals.

b) Adenoviral Vectors

The construction of replication-defective adenoviruses has beendescribed (Berkner et al., J. Virology 61:1213-1220 (1987); Massie etal., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et al., J. Virology57:267-274 (1986); Davidson et al., J. Virology 61:1226-1239 (1987);Zhang “Generation and identification of recombinant adenovirus byliposome-mediated transfection and PCR analysis” BioTechniques15:868-872 (1993)). The benefit of the use of these viruses as vectorsis that they are limited in the extent to which they can spread to othercell types, since they can replicate within an initial infected cell,but are unable to form new infectious viral particles. Recombinantadenoviruses have been shown to achieve high efficiency gene transferafter direct, in vivo delivery to airway epithelium, hepatocytes,vascular endothelium, CNS parenchyma and a number of other tissue sites(Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin.Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092(1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle, Science259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992);Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout,Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993);Caillaud, Eur. J. Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen.Virology 74:501-507 (1993)). Recombinant adenoviruses achieve genetransduction by binding to specific cell surface receptors, after whichthe virus is internalized by receptor-mediated endocytosis, in the samemanner as wild type or replication-defective adenovirus (Chardonnet andDales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985);Seth, et al., J. Virol. 51:650-655 (1984); Seth, et al., Mol. Cell.Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991);Wickham et al., Cell 73:309-319 (1993)).

A viral vector can be one based on an adenovirus which has had the E1gene removed and these virons are generated in a cell line such as thehuman 293 cell line. In another preferred embodiment both the E1 and E3genes are removed from the adenovirus genome.

c) Adeno-Associated Viral Vectors

Another type of viral vector is based on an adeno-associated virus(AAV). This defective parvovirus is a preferred vector because it caninfect many cell types and is nonpathogenic to humans. AAV type vectorscan transport about 4 to 5 kb and wild type AAV is known to stablyinsert into chromosome 19. Vectors which contain this site specificintegration property are preferred. An especially preferred embodimentof this type of vector is the P4.1 C vector produced by Avigen, SanFrancisco, Calif., which can contain the herpes simplex virus thymidinekinase gene, HSV-tk, and/or a marker gene, such as the gene encoding thegreen fluorescent protein, GFP.

In another type of AAV virus, the AAV contains a pair of invertedterminal repeats (ITRs) which flank at least one cassette containing apromoter which directs cell-specific expression operably linked to aheterologous gene. Heterologous in this context refers to any nucleotidesequence or gene which is not native to the AAV or B19 parvovirus.

Typically the AAV and B19 coding regions have been deleted, resulting ina safe, noncytotoxic vector. The AAV ITRs, or modifications thereof,confer infectivity and site-specific integration, but not cytotoxicity,and the promoter directs cell-specific expression. U.S. Pat. No.6,261,834 is herein incorporated by reference for material related tothe AAV vector.

The disclosed vectors thus provide DNA molecules which are capable ofintegration into a mammalian chromosome without substantial toxicity.

The inserted genes in viral and retroviral usually contain promoters,and/or enhancers to help control the expression of the desired geneproduct. A promoter is generally a sequence or sequences of DNA thatfunction when in a relatively fixed location in regard to thetranscription start site. A promoter contains core elements required forbasic interaction of RNA polymerase and transcription factors, and maycontain upstream elements and response elements.

d) Large Payload Viral Vectors

Molecular genetic experiments with large human herpesviruses haveprovided a means whereby large heterologous DNA fragments can be cloned,propagated and established in cells permissive for infection withherpesviruses (Sun et al., Nature genetics 8: 33-41, 1994; Cotter andRobertson,.Curr Opin Mol Ther 5: 633-644, 1999). These large DNA viruses(herpes simplex virus (HSV) and Epstein-Barr virus (EBV), have thepotential to deliver fragments of human heterologous DNA>150 kb tospecific cells. EBV recombinants can maintain large pieces of DNA in theinfected B-cells as episomal DNA. Individual clones carried humangenomic inserts up to 330 kb appeared genetically stable The maintenanceof these episomes requires a specific EBV nuclear protein, EBNA1,constitutively expressed during infection with EBV. Additionally, thesevectors can be used for transfection, where large amounts of protein canbe generated transiently in vitro. Herpesvirus amplicon systems are alsobeing used to package pieces of DNA>220 kb and to infect cells that canstably maintain DNA as episomes.

Other useful systems include, for example, replicating andhost-restricted non-replicating vaccinia virus vectors.

2. Non-Nucleic Acid Based Systems

The disclosed compositions can be delivered to the target cells in avariety of ways. For example, the compositions can be delivered throughelectroporation, or through lipofection, or through calcium phosphateprecipitation. The delivery mechanism chosen will depend in part on thetype of cell targeted and whether the delivery is occurring for examplein vivo or in vitro.

Thus, the compositions can comprise, in addition to the disclosedvectors, for example, lipids such as liposomes, such as cationicliposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes.Liposomes can further comprise proteins to facilitate targeting aparticular cell, if desired. Administration of a composition comprisinga compound and a cationic liposome can be administered to the bloodafferent to a target organ or inhaled into the respiratory tract totarget cells of the respiratory tract. Regarding liposomes, see, e.g.,Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989); Feigner etal. Proc. Nail. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat.No.4,897,355. Furthermore, the compound can be administered as acomponent of a microcapsule that can be targeted to specific cell types,such as macrophages, or where the diffusion of the compound or deliveryof the compound from the microcapsule is designed for a specific rate ordosage.

In the methods described above which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), delivery of the compositions to cells canbe via a variety of mechanisms. As one example, delivery can be via aliposome, using commercially available liposome preparations such asLIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.),SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (PromegaBiotec, Inc., Madison, Wis.), as well as other liposomes developedaccording to procedures standard in the art. In addition, the disclosednucleic acid or vector can be delivered in vivo by electroporation, thetechnology for which is available from Genetronics, Inc. (San Diego,Calif.) as well as by means of a SONOPORATION machine (ImaRxPharmaceutical Corp., Tucson, Ariz.).

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via antibodies, receptors, or receptor ligands. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter, et al., BioconjugateChem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer, 60:275-281,(1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, etal., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., CancerImmunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie,Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)). These techniques can be used for avariety of other specific cell types. Vehicles such as “stealth” andother antibody conjugated liposomes (including lipid mediated drugtargeting to colonic carcinoma), receptor mediated targeting of DNAthrough cell specific ligands, lymphocyte directed tumor targeting, andhighly specific therapeutic retroviral targeting of murine glioma cellsin vivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

Nucleic acids that are delivered to cells which are to be integratedinto the host cell genome, typically contain integration sequences.These sequences are often viral related sequences, particularly whenviral based systems are used. These viral intergration systems can alsobe incorporated into nucleic acids which are to be delivered using anon-nucleic acid based system of deliver, such as a liposome, so thatthe nucleic acid contained in the delivery system can be come integratedinto the host genome.

Other general techniques for integration into the host genome include,for example, systems designed to promote homologous recombination withthe host genome. These systems typically rely on sequence flanking thenucleic acid to be expressed that has enough homology with a targetsequence within the host cell genome that recombination between thevector nucleic acid and the target nucleic acid takes place, causing thedelivered nucleic acid to be integrated into the host genome. Thesesystems and the methods necessary to promote homologous recombinationare known to those of skill in the art.

3. In Vivo/Ex Vivo

As described above, the compositions can be administered in apharmaceutically acceptable carrier and can be delivered to thesubject=s cells in vivo and/or ex vivo by a variety of mechanisms wellknown in the art (e.g., uptake of naked DNA, liposome fusion,intramuscular injection of DNA via a gene gun, endocytosis and thelike).

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the body according to standard protocols well knownin the art. The compositions can be introduced into the cells via anygene transfer mechanism, such as, for example, calcium phosphatemediated gene delivery, electroporation, microinjection orproteoliposomes. The transduced cells can then be infused (e.g., in apharmaceutically acceptable carrier) or homotopically transplanted backinto the subject per standard methods for the cell or tissue type.Standard methods are known for transplantation or infusion of variouscells into a subject.

F. Expression Systems

The nucleic acids that are delivered to cells typically containexpression controlling systems. For example, the inserted genes in viraland retroviral systems usually contain promoters, and/or enhancers tohelp control the expression of the desired gene product. A promoter isgenerally a sequence or sequences of DNA that function when in arelatively fixed location in regard to the transcription start site. Apromoter contains core elements required for basic interaction of RNApolymerase and transcription factors, and may contain upstream elementsand response elements.

1. Viral Promoters and Enhancers

Preferred promoters controlling transcription from vectors in mammalianhost cells may be obtained from various sources, for example, thegenomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis-B virus and most preferably cytomegalovirus, orfrom heterologous mammalian promoters, e.g. beta actin promoter. Theearly and late promoters of the SV40 virus are conveniently obtained asan SV40 restriction fragment which also contains the SV40 viral originof replication (Fiers et al., Nature, 273: 113 (1978)). The immediateearly promoter of the human cytomegalovirus is conveniently obtained asa HindIII E restriction fragment (Greenway, P. J. et al., Gene 18:355-360 (1982)). Of course, promoters from the host cell or relatedspecies also are useful herein.

Enhancer generally refers to a sequence of DNA that functions at nofixed distance from the transcription start site and can be either 5′(Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3′(Lusky, M. L., et al., Mol. Cell Bio. 3: 1108 (1983)) to thetranscription unit. Furthermore, enhancers can be within an intron(Banerji, J. L. et al., Cell 33: 729 (1983)) as well as within thecoding sequence itself (Osborne, T. F., et al., Mol. Cell Bio. 4: 1293(1984)). They are usually between 10 and 300 bp in length, and theyfunction in cis. Enhancers f unction to increase transcription fromnearby promoters. Enhancers also often contain response elements thatmediate the regulation of transcription. Promoters can also containresponse elements that mediate the regulation of transcription.Enhancers often determine the regulation of expression of a gene. Whilemany enhancer sequences are now known from mammalian genes (globin,elastase, albumin, -fetoprotein and insulin), typically one will use anenhancer from a eukaryotic cell virus for general expression. Preferredexamples are the SV40 enhancer on the late side of the replicationorigin (bp 100-270), the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

The promotor and/or enhancer may be specifically activated either bylight or specific chemical events which trigger their function. Systemscan be regulated by reagents such as tetracycline and dexamethasone.There are also ways to enhance viral vector gene expression by exposureto irradiation, such as gamma irradiation, or alkylating chemotherapydrugs.

In certain embodiments the promoter and/or enhancer region can act as aconstitutive promoter and/or enhancer to maximize expression of theregion of the transcription unit to be transcribed. In certainconstructs the promoter and/or enhancer region be active in alleukaryotic cell types, even if it is only expressed in a particular typeof cell at a particular time. A preferred promoter of this type is theCMV promoter (650 bases). Other preferred promoters are SV40 promoters,cytomegalovirus (full length promoter), and retroviral vector LTR.

It has been shown that all specific regulatory elements can be clonedand used to construct expression vectors that are selectively expressedin specific cell types such as melanoma cells. The glial fibrillaryacetic protein (GFAP) promoter has been used to selectively expressgenes in cells of glial origin.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells) may also contain sequencesnecessary for the termination of transcription which may affect mRNAexpression. These regions are transcribed as polyadenylated segments inthe untranslated portion of the mRNA encoding tissue factor protein. The3′ untranslated regions also include transcription termination sites. Itis preferred that the transcription unit also contains a polyadenylationregion. One benefit of this region is that it increases the likelihoodthat the transcribed unit will be processed and transported like mRNA.The identification and use of polyadenylation signals in expressionconstructs is well established. It is preferred that homologouspolyadenylation signals be used in the transgene constructs. In certaintranscription units, the polyadenylation region is derived from the SV40early polyadenylation signal and consists of about 400 bases. It is alsopreferred that the transcribed units contain other standard sequencesalone or in combination with the above sequences improve expressionfrom, or stability of, the construct.

2. Markers

The viral vectors can include nucleic acid sequence encoding a markerproduct. This marker product is used to determine if the gene has beendelivered to the cell and once delivered is being expressed. Preferredmarker genes are the E. Coli lacZ gene, which encodes β-galactosidase,and green fluorescent protein.

In some embodiments the marker may be a selectable marker. Examples ofsuitable selectable markers for mammalian cells are dihydrofolatereductase (DHFR), thymidine kinase, neomycin, neomycin analog G418,hydromycin, and puromycin. When such selectable markers are successfullytransferred into a mammalian host cell, the transformed mammalian hostcell can survive if placed under selective pressure. There are twowidely used distinct categories of selective regimes. The first categoryis based on a cell's metabolism and the use of a mutant cell line whichlacks the ability to grow independent of a supplemented media. Twoexamples are: CHO DHFR-cells and mouse LTK-cells. These cells lack theability to grow without the addition of such nutrients as thymidine orhypoxanthine. Because these cells lack certain genes necessary for acomplete nucleotide synthesis pathway, they cannot survive unless themissing nucleotides are provided in a supplemented media. An alternativeto supplementing the media is to introduce an intact DHFR or TK geneinto cells lacking the respective genes, thus altering their growthrequirements. Individual cells which were not transformed with the DHFRor TK gene will not be capable of survival in non-supplemented media.

The second category is dominant selection which refers to a selectionscheme used in any cell type and does not require the use of a mutantcell line. These schemes typically use a drug to arrest growth of a hostcell. Those cells which have a novel gene would express a proteinconveying drug resistance and would survive the selection. Examples ofsuch dominant selection use the drugs neomycin, (Southern P. and Berg,P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan,R. C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B.et al., Mol. Cell. Biol. 5: 410-413 (1985)). The three examples employbacterial genes under eukaryotic control to convey resistance to theappropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid)or hygromycin, respectively. Others include the neomycin analog G418 andpuramycin.

G. Pharmaceutical Carriers/Delivery of Pharamceutical Products

As described above, the compositions can also be administered in vivo ina pharmaceutically acceptable carrier. By “pharmaceutically acceptable”is meant a material that is not biologically or otherwise undesirable,i.e., the material may be administered to a subject, along with thenucleic acid or vector, without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.The carrier would naturally be selected to minimize any degradation ofthe active ingredient and to minimize any adverse side effects in thesubject, as would be well known to one of skill in the art.

The compositions may be administered orally, parenterally (e.g.,intravenously), by intramuscular injection, by intraperitonealinjection, transdermally, extracorporeally, topically or the like,including topical intranasal administration or administration byinhalant. As used herein, “topical intranasal administration” meansdelivery of the compositions into the nose and nasal passages throughone or both of the nares and can comprise delivery by a sprayingmechanism or droplet mechanism, or through aerosolization of the nucleicacid or vector. Administration of the compositions by inhalant can bethrough the nose or mouth via delivery by a spraying or dropletmechanism. Delivery can also be directly to any area of the respiratorysystem (e.g., lungs) via intubation. The exact amount of thecompositions required will vary from subject to subject, depending onthe species, age, weight and general condition of the subject, theseverity of the allergic disorder being treated, the particular nucleicacid or vector used, its mode of administration and the like. Thus, itis not possible to specify an exact amount for every composition.However, an appropriate amount can be determined by one of ordinaryskill in the art using only routine experimentation given the teachingsherein.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein.

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via antibodies, receptors, or receptor ligands. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter, et al., BioconjugateChem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer, 60:275-281,(1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, etal., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., CancerImmunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie,Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)). Vehicles such as “stealth” and otherantibody conjugated liposomes (including lipid mediated drug targetingto colonic carcinoma), receptor mediated targeting of DNA through cellspecific ligands, lymphocyte directed tumor targeting, and highlyspecific therapeutic retroviral targeting of murine glioma cells invivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

1. Pharmaceutically Acceptable Carriers

The compositions, including antibodies, can be used therapeutically incombination with a pharmaceutically acceptable carrier.

Suitable carriers and their formulations are described in Remington: TheScience and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, MackPublishing Company, Easton, Pa. 1995. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include, but are not limited to,saline, Ringer's solution and dextrose solution. The pH of the solutionis preferably from about 5 to about 8, and more preferably from about 7to about 7.5. Further carriers include sustained release preparationssuch as semipermeable matrices of solid hydrophobic polymers containingthe antibody, which matrices are in the form of shaped articles, e.g.,films, liposomes or microparticles. It will be apparent to those personsskilled in the art that certain carriers may be more preferabledepending upon, for instance, the route of administration andconcentration of composition being administered.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.

Examples of materials which can serve as pharmaceutically acceptablecarriers include sugars, such as lactose, glucose and sucrose; starches,such as corn starch and potato starch; cellulose, and its derivatives,such as sodium carboxymethyl cellulose, ethyl cellulose and celluloseacetate; powdered tragacanth; malt; gelatin; talc; excipients, such ascocoa butter and suppository waxes; oils, such as peanut oil, cottonseedoil, safflower oil, sesame oil, olive oil, corn oil and soybean oil;glycols, such as propylene glycol; polyols, such as glycerin, sorbitol,mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyllaurate; agar; buffering agents, such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters,polycarbonates and/or polyanhydrides ; and other non-toxic compatiblesubstances employed in pharmaceutical formulations. Wetting agents,emulsifiers and lubricants, such as sodium lauryl sulfate and magnesiumstearate, as well as coloring agents, release agents, coating agents,sweetening, flavoring and perfuming agents, preservatives andantioxidants can also be present in the compositions. The compositionsof the present invention can be administered parenterally (for example,by intravenous, intraperitoneal, subcutaneous or intramuscularinjection), topically (including buccal and sublingual), orally,intranasally, intravaginally, or rectally according to standard medicalpractices.

Pharmaceutical compositions may include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions may also includeone or more active ingredients such as antimicrobial agents,antiinflammatory agents, anesthetics, and the like.

The pharmaceutical composition may be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. Administration may be topically (includingophthalmically, vaginally, rectally, intranasally), orally, byinhalation, or parenterally, for example by intravenous drip,subcutaneous, intraperitoneal or intramuscular injection. The disclosedantibodies can be administered intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

Pharmaceutical compositions can be in the form of pharmaceuticallyacceptable salts and complexes and can be provided in a pharmaceuticallyacceptable carrier and at an appropriate dose. Such pharmaceuticalcompositions can be prepared by methods and contain carriers which arewell known in the art. A generally recognized compendium of such methodsand ingredients is Remington: The Science and Practice of Pharmacy,Alfonso R. Gennaro, editor, 20th ed. Lippincott Williams & Wilkins:Philadelphia, Pa., 2000. A pharmaceutically-acceptable carrier,composition or vehicle, such as a liquid or solid filler, diluent,excipient, or solvent encapsulating material, is involved in carrying ortransporting the subject compound from one organ, or portion of thebody, to another organ, or portion of the body. Each carrier must beacceptable in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion or metabolism of the particularcompound being employed, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompound employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell known in the medical arts.

As will be understood by those of skill in the art upon reading thisdisclosure, additional compounds to those exemplified herein can beidentified routinely in accordance with the screening methods taughtherein. Additional compounds for screening can be selected randomly byone skilled in the art, based upon computational prediction, and/orbased upon their containing a structure of Formula I, II or III or astructure similar to that of the exemplary compounds disclosed herein.

2. Therapeutic Uses

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of a compound at levels lower than that required in order toachieve the desired therapeutic effect and gradually increase the dosageuntil the desired effect is achieved. This is considered to be withinthe skill of the artisan and one can review the existing literature on aspecific compound or similar compounds to determine optimal dosing.

Effective dosages and schedules for administering the compositions maybe determined empirically, and making such determinations is within theskill in the art. The dosage ranges for the administration of thecompositions are those large enough to produce the desired effect inwhich the symptoms of the disorder are effected. The dosage should notbe so large as to cause adverse side effects, such as unwantedcross-reactions, anaphylactic reactions, and the like. Generally, thedosage will vary with the age, condition, sex and extent of the diseasein the patient, route of administration, or whether other drugs areincluded in the regimen, and can be determined by one of skill in theart. The dosage can be adjusted by the individual physician in the eventof any counterindications. Dosage can vary, and can be administered inone or more dose administrations daily, for one or several days.Guidance can be found in the literature for appropriate dosages forgiven classes of pharmaceutical products. For example, guidance inselecting appropriate doses for antibodies can be found in theliterature on therapeutic uses of antibodies, e.g., Handbook ofMonoclonal Antibodies, Ferrone et al., eds., Noges Publications, ParkRidge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies inHuman Diagnosis and Therapy, Haber et al., eds., Raven Press, New York(1977) pp. 365-389. A typical daily dosage of the antibody used alonemight range from about 1 μg/kg to up to 100 mg/kg of body weight or moreper day, depending on the factors mentioned above.

H. Compositions Identified by Screening with DisclosedCompositions/Combinatorial Chemistry

1. Combinatorial Chemistry

The disclosed compositions can be used as targets for any combinatorialtechnique to identify molecules or macromolecular molecules thatinteract with the disclosed compositions in a desired way. The nucleicacids, peptides, and related molecules disclosed herein can be used astargets for the combinatorial approaches. For example, SIGK can be usedto identify and agent that will disrupt the binding of SIGK to theprotein interaction site of Gβ. Also disclosed are the compositions thatare identified through combinatorial techniques or screening techniquesdisclosed herein, such as, for example, M119.

It is understood that when using the disclosed compositions incombinatorial techniques or screening methods, molecules, such asmacromolecular molecules, will be identified that have particulardesired properties such as inhibition or stimulation or the targetmolecule's function. The molecules identified and isolated when usingthe disclosed compositions, such as, M119, are also disclosed. Thus, theproducts produced using the combinatorial or screening approaches thatinvolve the disclosed compositions, such as, M119, are also consideredherein disclosed.

It is understood that the disclosed methods for identifying moleculesthat inhibit the interactions between, for example, Gβγ and bARK1 or Gβγand SIGK can be performed using high through put means. For example,putative inhibitors can be identified using Fluorescence ResonanceEnergy Transfer (FRET) to quickly identify interactions. The underlyingtheory of the techniques is that when two molecules are close in space,ie, interacting at a level beyond background, a signal is produced or asignal can be quenched. Then, a variety of experiments can be performed,including, for example, adding in a putative inhibitor. If the inhibitorcompetes with the interaction between the two signaling molecules, thesignals will be removed from each other in space, and this will cause adecrease or an increase in the signal, depending on the type of signalused. This decrease or increasing signal can be correlated to thepresence or absence of the putative inhibitor. Any signaling means canbe used. For example, disclosed are methods of identifying an inhibitorof the interaction between any two of the disclosed moleculescomprising, contacting a first molecule and a second molecule togetherin the presence of a putative inhibitor, wherein the first molecule orsecond molecule comprises a fluorescence donor, wherein the first orsecond molecule, typically the molecule not comprising the donor,comprises a fluorescence acceptor; and measuring Fluorescence ResonanceEnergy Transfer (FRET), in the presence of the putative inhibitor andthe in absence of the putative inhibitor, wherein a decrease in FRET inthe presence of the putative inhibitor as compared to FRET measurementin its absence indicates the putative inhibitor inhibits binding betweenthe two molecules. This type of method can be performed with a cellsystem as well.

Combinatorial chemistry includes but is not limited to all methods forisolating small molecules or macromolecules that are capable of bindingeither a small molecule or another macromolecule, typically in aniterative process. Proteins, oligonucleotides, and sugars are examplesof macromolecules. For example, oligonucleotide molecules with a givenfunction, catalytic or ligand-binding, can be isolated from a complexmixture of random oligonucleotides in what has been referred to as “invitro genetics” (Szostak, TIBS 19:89, 1992). One synthesizes a largepool of molecules bearing random and defined sequences and subjects thatcomplex mixture, for example, approximately 10¹⁵ individual sequences in100 μg of a 100 nucleotide RNA, to some selection and enrichmentprocess. Through repeated cycles of affinity chromatography and PCRamplification of the molecules bound to the ligand on the column,Ellington and Szostak (1990) estimated that 1 in 10¹⁰ RNA moleculesfolded in such a way as to bind a small molecule dyes. DNA moleculeswith such ligand-binding behavior have been isolated as well (Ellingtonand Szostak, 1992; Bock et al, 1992). Techniques aimed at similar goalsexist for small organic molecules, proteins, antibodies and othermacromolecules known to those of skill in the art. Screening sets ofmolecules for a desired activity whether based on small organiclibraries, oligonucleotides, or antibodies is broadly referred to ascombinatorial chemistry. Combinatorial techniques are particularlysuited for defining binding interactions between molecules and forisolating molecules that have a specific binding activity, often calledaptamers when the macromolecules are nucleic acids.

There are a number of methods for isolating proteins which either havede novo activity or a modified activity. For example, phage displaylibraries have been used to isolate numerous peptides that interact witha specific target. (See for example, U.S. Pat. Nos. 6,031,071;5,824,520; 5,596,079; and 5,565,332 which are herein incorporated byreference at least for their material related to phage display andmethods relate to combinatorial chemistry)

A preferred method for isolating proteins that have a given function isdescribed by Roberts and Szostak (Roberts R. W. and Szostak J. W. Proc.Natl. Acad. Sci. USA, 94(23)12997-302 (1997). This combinatorialchemistry method couples the functional power of proteins and thegenetic power of nucleic acids. An RNA molecule is generated in which apuromycin molecule is covalently attached to the 3′-end of the RNAmolecule. An in vitro translation of this modified RNA molecule causesthe correct protein, encoded by the RNA to be translated. In addition,because of the attachment of the puromycin, a peptdyl acceptor whichcannot be extended, the growing peptide chain is attached to thepuromycin which is attached to the RNA. Thus, the protein molecule isattached to the genetic material that encodes it. Normal in vitroselection procedures can now be done to isolate functional peptides.Once the selection procedure for peptide function is completetraditional nucleic acid manipulation procedures are performed toamplify the nucleic acid that codes for the selected functionalpeptides. After amplification of the genetic material, new RNA istranscribed with puromycin at the 3′-end, new peptide is translated andanother functional round of selection is performed. Thus, proteinselection can be performed in an iterative manner just like nucleic acidselection techniques. The peptide which is translated is controlled bythe sequence of the RNA attached to the puromycin. This sequence can beanything from a random sequence engineered for optimum translation (i.e.no stop codons etc.) or it can be a degenerate sequence of a known RNAmolecule to look for improved or altered function of a known peptide.The conditions for nucleic acid amplification and in vitro translationare well known to those of ordinary skill in the art and are preferablyperformed as in Roberts and Szostak (Roberts R. W. and Szostak J. W.Proc. Natl. Acad. Sci. USA, 94(23)12997-302 (1997)).

Another preferred method for combinatorial methods designed to isolatepeptides is described in Cohen et al. (Cohen B. A., et al., Proc. Natl.Acad. Sci. USA 95(24):14272-7 (1998)). This method utilizes and modifiestwo-hybrid technology. Yeast two-hybrid systems are useful for thedetection and analysis of protein:protein interactions. The two-hybridsystem, initially described in the yeast Saccharomyces cerevisiae, is apowerful molecular genetic technique for identifying new regulatorymolecules, specific to the protein of interest (Fields and Song, Nature340:245-6 (1989)). Cohen et al., modified this technology so that novelinteractions between synthetic or engineered peptide sequences could beidentified which bind a molecule of choice. The benefit of this type oftechnology is that the selection is done in an intracellularenvironment. The method utilizes a library of peptide molecules thatattached to an acidic activation domain.

Using methodology well known to those of skill in the art, incombination with various combinatorial libraries, one can isolate andcharacterize those small molecules or macromolecules, which bind to orinteract with the desired target. The relative binding affinity of thesecompounds can be compared and optimum compounds identified usingcompetitive binding studies, which are well known to those of skill inthe art.

Techniques for making combinatorial libraries and screeningcombinatorial libraries to isolate molecules which bind a desired targetare well known to those of skill in the art. Representative techniquesand methods can be found in but are not limited to U.S. Pat. Nos.5,084,824, 5,288,514, 5,449,754, 5,506,337, 5,539,083, 5,545,568,5,556,762, 5,565,324, 5,565,332, 5,573,905, 5,618,825, 5,619,680,5,627,210, 5,646,285, 5,663,046, 5,670,326, 5,677,195, 5,683,899,5,688,696, 5,688,997, 5,698,685, 5,712,146, 5,721,099, 5,723,598,5,741,713, 5,792,431, 5,807,683, 5,807,754, 5,821,130, 5,831,014,5,834,195, 5,834,318, 5,834,588, 5,840,500, 5,847,150, 5,856,107,5,856,496, 5,859,190, 5,864,010, 5,874,443, 5,877,214, 5,880,972,5,886,126, 5,886,127, 5,891,737, 5,916,899, 5,919,955, 5,925,527,5,939,268, 5,942,387, 5,945,070, 5,948,696, 5,958,702, 5,958,792,5,962,337, 5,965,719, 5,972,719, 5,976,894, 5,980,704, 5,985,356,5,999,086, 6,001,579, 6,004,617, 6,008,321, 6,017,768, 6,025,371,6,030,917, 6,040,193, 6,045,671, 6,045,755, 6,060,596, and 6,061,636.

Combinatorial libraries can be made from a wide array of molecules usinga number of different synthetic techniques. For example, librariescontaining fused 2,4-pyrimidinediones (U.S. Pat. No. 6,025,371)dihydrobenzopyrans (U.S. Pat. Nos. 6,017,768and 5,821,130), amidealcohols (U.S. Pat. No. 5,976,894), hydroxy-amino acid amides (U.S. Pat.No. 5,972,719) carbohydrates (U.S. Pat. No. 5,965,719),1,4-benzodiazepin-2,5-diones (U.S. Pat. No. 5,962,337), cyclics (U.S.Pat. No. 5,958,792), biaryl amino acid amides (U.S. Pat. No. 5,948,696),thiophenes (U.S. Pat. No. 5,942,387), tricyclic Tetrahydroquinolines(U.S. Pat. No. 5,925,527), benzofurans (U.S. Pat. No. 5,919,955),isoquinolines (U.S. Pat. No. 5,916,899), hydantoin and thiohydantoin(U.S. Pat. No. 5,859,190), indoles (U.S. Pat. No. 5,856,496),imidazol-pyrido-indole and imidazol-pyrido-benzothiophenes (U.S. Pat.No. 5,856,107) substituted 2-methylene-2,3-dihydrothiazoles (U.S. Pat.No. 5,847,150), quinolines (U.S. Pat. No. 5,840,500), PNA (U.S. Pat. No.5,831,014), containing tags (U.S. Pat. No. 5,721,099), polyketides (U.S.Pat. No. 5,712,146), morpholino-subunits (U.S. Pat. Nos. 5,698,685 and5,506,337), sulfamides (U.S. Pat. No. 5,618,825), and benzodiazepines(U.S. Pat. No. 5,288,514).

As used herein combinatorial methods and libraries included traditionalscreening methods and libraries as well as methods and libraries used ininterative processes.

2. Computer Assisted Drug Design

The disclosed compositions can be used as targets for any molecularmodeling technique to identify either the structure of the disclosedcompositions or to identify potential or actual molecules, such as smallmolecules, which interact in a desired way with the disclosedcompositions. The nucleic acids, peptides, and related moleculesdisclosed herein can be used as targets in any molecular modelingprogram or approach.

It is understood that when using the disclosed compositions in modelingtechniques, molecules, such as macromolecular molecules, will beidentified that have particular desired properties such as inhibition orstimulation of Gβγ function. The molecules identified and isolated whenusing the disclosed compositions, such as, M119, are also disclosed.Thus, the products produced using the molecular modeling approaches thatinvolve the disclosed compositions, such as, M119, are also consideredherein disclosed.

Thus, one way to isolate molecules that bind a molecule of choice isthrough rational design. This is achieved through structural informationand computer modeling. Computer modeling technology allows visualizationof the three-dimensional atomic structure of a selected molecule and therational design of new compounds that will interact with the molecule.The three-dimensional construct typically depends on data from x-raycrystallographic analyses or NMR imaging of the selected molecule. Themolecular dynamics require force field data. The computer graphicssystems enable prediction of how a new compound will link to the targetmolecule and allow experimental manipulation of the structures of thecompound and target molecule to perfect binding specificity. Predictionof what the molecule-compound interaction will be when small changes aremade in one or both requires molecular mechanics software andcomputationally intensive computers, usually coupled with user-friendly,menu-driven interfaces between the molecular design program and theuser.

Examples of molecular modeling systems are the CHARMm and QUANTAprograms, Polygen Corporation, Waltham, Mass. CHARMm performs the energyminimization and molecular dynamics functions. QUANTA performs theconstruction, graphic modeling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

A number of articles review computer modeling of drugs interactive withspecific proteins, such as Rotivinen, et al., 1988 Acta PharmaceuticaFennica 97, 159-166; Ripka, New Scientist 54-57 (Jun. 16, 1988);McKinaly and Rossmann, 1989 Annu. Rev. Pharmacol. _(—) Toxiciol. 29,111-122; Perry and Davies, QSAR: Quantitative Structure-ActivityRelationships in Drug Design pp. 189-193 (Alan R. Liss, Inc. 1989);Lewis and Dean, 1989 Proc. R. Soc. Lond. 236, 125-140 and 141-162; and,with respect to a model enzyme for nucleic acid components, Askew, etal., 1989 J. Am. Chem. Soc. 111, 1082-1090. Other computer programs thatscreen and graphically depict chemicals are available from companiessuch as BioDesign, Inc., Pasadena, Calif., Allelix, Inc, Mississauga,Ontario, Canada, and Hypercube, Inc., Cambridge, Ontario. Although theseare primarily designed for application to drugs specific to particularproteins, they can be adapted to design of molecules specificallyinteracting with specific regions of DNA or RNA, once that region isidentified.

Although described above with reference to design and generation ofcompounds which could alter binding, one could also screen libraries ofknown compounds, including natural products or synthetic chemicals, andbiologically active materials, including proteins, for compounds whichalter substrate binding or enzymatic activity.

I. Kits

Disclosed herein are kits that are drawn to reagents that can be used inpracticing the methods disclosed herein. The kits can include anyreagent or combination of reagent discussed herein or that would beunderstood to be required or beneficial in the practice of the disclosedmethods. For example, the kits could include primers to perform theamplification reactions discussed in certain embodiments of the methods,as well as the buffers and enzymes required to use the primers asintended. Thus, for example, disclosed herein are kits for identifyingan agent that binds at least one amino acid residue of the proteininteraction site of the β subunit is also provided. The kit of theinvention contains a SIGK peptide or SIGK peptide derivative.

J. Compositions with Similar Functions

It is understood that the compositions disclosed herein have certainfunctions, such as inhibiting Gβγ activity or binding the proteininteraction site of Gβ. Disclosed herein are certain structuralrequirements for performing the disclosed functions, and it isunderstood that there are a variety of structures which can perform thesame function which are related to the disclosed structures, and thatthese structures will ultimately achieve the same result, for examplestimulation or inhibition Gβγ activity.

K. EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary and arenot intended to limit the disclosure. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.), butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

1. Example 1 Differential Targeting of Gβγ-Subunit Signaling with SmallMolecules

a) Materials and Methods

Peptides were purchased from Alpha Diagnostic International (SanAntonio, Tex.) or SIGMA®-Genosys (St. Louis, Mo.), HPLC purified togreater than 90% and masses confirmed by mass spectroscopy. Ni-NTAagarose was from QIAGEN® (Valencia, Calif.). Streptavidin-coatedpoly-styrene beads were from Spherotec (Libertyville, Ill.).HRP-conjugated anti-M13 antibody was from Amersham Biosciences(Piscataway, N.J.). HRP-conjugated Neutravidin was from Pierce(Rockford, Ill.). All molecular biology reagents were from INVITROGEN™(Carlsbad, Calif.) unless otherwise indicated.

(1) Expression and Purification of Gβ1Y₂ and SIGK Peptide

Baculoviruses harboring cDNA for wild-type bovine Gβ1 and N-terminally(His) _(ε)-tagged bovine Gγ₂ were used to produce proteins of the same.High 5 cells (INVITROGEN™, Carlsbad, Calif.; 2×1O⁶ cells/mL) wereinfected with high titer Gβ1 and Gγ₂ baculoviruses. Gβ1γ₂ was purifiedaccording to standard methods (Kozaza and Gilman (1995) J. Biol. Chem.270:1734-41), with modifications. All steps were carried out at 4° C.Cells were harvested 60 hours post-infection by centrifugation at 260Og, then resuspended in 50 mL of lysis buffer (20 rtiM HEPES, pH 8, 150mM NaCl, 5 mM β-ME, 1 mM EDTA, 1 mL SIGMA® protease inhibitor cocktailP-2714) per liter of cell culture. Cells were lysed by sonication andcentrifuged at 2600 gr to pellet the membranes. Resuspension andhomogenization of membranes was accomplished by douncing in 100 mL lysisbuffer. The membranes were solubilized by adding 1% Lubrol (C12E10,SIGMA®, St. Louis, Mo.) with stirring and the resultant solutionclarified by ultracentrifugation at 125,000^. The supernatant was loadedonto Ni-NTA agarose (QIAGEN®, Valencia, Calif.) equilibrated with lysisbuffer+1% Lubrol. The column was washed and the Lubrol exchanged forsodium cholate using buffers Ni-A (20 mM HEPES, pH 8, 0.4 M NaCl, 5 mMβ-ME, 0.5% Lubrol, 0.15% cholate) and Ni—B (20 mM HEPES pH 8 , 0.1 MNaCl, 5 mM β-ME, 0.25% Lubrol, 0.3% cholate). Gβ1γ2 eluted in Ni—C (20mM HEPES pH 8 , 0.01 M NaCl, 5 mM β-ME, 1% cholate, 200 mM imidazole).The eluate was loaded onto a HITRAP™ Q (Amersham Biosciences,Piscataway, N.J.) column pre-equilibrated with QA (20 mM HEPES, pH 8 , 5mM β-ME, 0.7% CHAPS, 1 mM EDTA). Gβ1γ₂ eluted in a gradient using QB(QA+1.0 M NaCl). Fractions containing Gβ1γ₂ were analyzed by SDS-PAGEand pooled. Gel filtration was performed using a tandem SEPHADEX®75:SEPHADEX® 200 column (Amersham Biosciences, Piscataway, N.J.)equilibrated with buffer GF+CHAPS (20 mM HEPES, pH 8, 150 mM NaCl, 10 mMβ-ME, 1 mM EDTA, 0.7% CHAPS). The purified yield was typically 1 mgGβ1γ₂ per liter of cell culture.

SIGK peptide(Ser-Ile-Gly-Lys-Ala-Phe-Lys-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp; SEQ ID NO:2) was synthesized using well-established methods. No modifications weremade to the peptide termini; purification was by reverse phase-HPLCchromatography on a VYDAC® C4 semi-preparative column.

(2) Crystallography

SIGK peptide was added to β1γ₂ in 1.5 molar excess, and the Gβ1γ₂<<SIGKcomplex was used at 7 mg/mL for crystallization. Crystals were grown byvapor diffusion using equal volumes (2 μL) of protein and reservoirsolution (15-17% PEG 4000, 100 mM HEPES, pH 7.5, 0.01-0.05 M Na-Acetate,10% glycerol) at 20° C. Crystals attained dimensions of 150 μm×50 μm×20μm within one week. Crystals were cryoprotected in 15% glycerol andfrozen in liquid nitrogen.

Native crystals of Gβ1γ₂*SIGK were screened at Advanced Light Source(ALS) beamlines 8.2.1 and 8.2.2 (Berkeley, Calif.) and at the AdvancedPhoton Source (APS) beamline BM-19 (Chicago, Ill.). A dataset from ALS8.2.2 was used to determine the structure. Over 100 crystals werescreened; diffraction limits varied from 7A to the 2.7A dataset used forstructure determination. Diffraction data were indexed, integrated, andscaled using the software package HKL2000

(Otwinowski and Minor (1997) In: Methods in Enzymology, Vol.276:307-326) (Table 10). The space-group of the crystals was P2i2i2i.

TABLE 10 Data Collection Space Group P2₁2₁2₁ Unique Reflections 9729Unit Cell Redundancy ¹ 3.5 (1.8) a (Å) 45.468 Completeness (%) ¹ 90.1(56.2) b 74.669 <1/σ> ¹ 13.5 (1.6)  c 108.023 Rsym ^(1, 2)  8.7 (41.4) α(°) 90 Mosaicity (°) 2.3 β 90 Wilson B-factor (Å) 61.8 γ 90 D_(min) (Å)2.7 L. Refinement Resolution (Å) 45.4-2.7 R.m.s Deviations Number ofatoms ³ Bond lengths (Å) 0.006 Protein Bond Angles (°) 1.3 WaterR_(work) (%) ⁴ R.m.s. B factors (Å²) R_(free) (%) ⁵ Bonded main chain1.29 Bonded side chain 18.1 Average B-factor (Å) ⁶ 46.3 The final modelcontains residues 2-340 of Gβ1 (of 340), 7-52 of Gγ₂ (of 68), 1-13 ofSIGK (of 15), and 37 water molecules. ¹ Numbers in parenthesescorrespond to the highest resolution shell, 2.8-2.7 A. ² R_(sym) = Σ_(h)Σi |li(h) − <I(h)>|/Σ_(h) Σi Ii (h), where Ii (h) and <I (h)> are thei^(th) and mean measurement of the intensity of reflection h,respectively. ³ The final model contains residues 2-340 of Gβ1 (of 340),7-52 of Gγ₂ (of 68), and 1-13 of SIGK (of 15). ⁴ Rwork = Σ_(h) I I F₀(h) I − |F_(c)(h)∥/Σ_(h) |F_(o)(h)|, where F₀ (h) and F_(c) (h) are theobserved and calculated structure factors, respectively. An J/σ cutoffwas not used in the final calculations of R-factors. ⁵ Rfree is theR-factor obtained for a test set of reflections consisting of a randomlyselected 8% of the data. ⁶ B-factors at the N-termini, including Gβ1residues 2-41 and Gγ₂ residues 7-13, are greater than 80 A².

The structure of the Gβ1γ₂*SIGK complex was solved by the molecularreplacement method using the program PHASER (Storoni, et al. (2004) ActaCrystallogr. D Biol. Crystallogr. 60:432-8; Read (2001) ActaCrystallogr. D Biol. Crystallogr. 57:1373-82). The coordinates of Gβ1γ₂in the Gβχγ₂>>GRK2 complex (10 MW, 100% sequence identity) were used asthe search model. After rigid body refinement using the maximumlikelihood minimization target in CNS version 1.1 (Adams, et al. (1997)Proc. Natl. Acad. Sci. USA 94:5018-23; Brunger, et al. (1998) ActaCrystallographica Section D 54:905-921), the model was further refinedby using a combination of simulated annealing, Powell minimization, andB factor refinement. The sigma A-weighted 2Fo-Fc electron density mapcomputed with refined phases revealed clear main chain density for tenresidues of the SIGK peptide along with identifiable side chain densityfor several SIGK residues. Subsequent model building was performed in O(Jones, et al. (1991) Acta Crystallographica Section A 47:110-119)followed by simulated annealing, energy minimization, and B factorrefinement using CNS. PROCHECK (Laskowski, et al. (1993) J. Appl.Crystallography 26:283-291) analysis indicates that all residues exhibitmain chain conformations in most favored or additional allowed regionsof φ, ψ space (Table 10). Calculations of surface accessibility,Gβ1γ₂<<SIGK contacts and RMSD between structures were carried out usingprograms in the CNS suite.

(1) Construction and Partial Purification of Biotinylated Gβ1γ₂ (b-βγ)and b-βγ Mutants

Wild-type Gβ1 and Gp₁ mutants were made in the baculovirus vector PDW464which encodes a biotinylation site at a lysine upstream of the aminoterminus of Gβ1 (Goubaeva, et al. (2003) supra). Mutants were generatedby overlap extension PCR using standard protocols. The wild-type andmutant Gβ1 constructs consisted of a 20 amino acid biotin acceptorpeptide (BAP) sequence fused in-frame with the amino-terminus of rat Gβ1subunit. When coexpressed with biotin holoenzyme synthetase (BirA) inSf9 cells, the Gβ1 subunit becomes covalently biotinylated in vivo atthe specific lysine acceptor residue in the BAP. Using this approach,1-2 mg protein of purified protein can be obtained per liter of Sf9insect cells. As 45 ng of protein is used in the phage ELISA assay, asingle purification is sufficient for 10,000 to 30,000 binding assays.

Baculoviruses were generated via the BAC-TO-BAC® system following themanufacturer's instructions (GIBCO/BRL, Gaithersburg, Md.). Sf9 cells(200 tnL) were triply infected with 0.5 mL baculovirus encoding (His)6-Gαu, 4 mL of Gγ₂ virus, and 4 mL of either wild-type or mutated Gβ1virus. Gβ1γ₂ dimers were purified 60 hours postinfection using awell-established method with modifications as indicated (Kozasa andGilman (1995) supra). Cell pellets were lysed in 4 mL lysis buffer (50mM HEPES, pH 8.0, 3 πiM MgCl₂, 10 mM β-mercaptoethanol, 1 mM EDTA, 100mM NaCl, 10 μM GDP, and protease inhibitors) by four freeze-thaw cyclesin liquid nitrogen. Membranes were solubilized using 1% sodium cholate,clarified by ultracentrifugation at 100,00 Og for 20 minutes, dilutedinto buffer containing 0.5% lubrol , and mixed with Ni-NTA resin. Afterwashing thoroughly, Gβ1γ₂ subunits were eluted from bound Gαii by mixingbeads with buffer containing 50 mM MgCl₂, 10 mM NaF, 10 μM AlCl₃, 1%cholate, and 5 mM imidazole at room temperature for one hour. Theconcentrations of b-βγ and b-βγ mutants were analyzed by comparativeimmunoblotting and chemiluminescence. Proteins were separated bySDS-PAGE, transferred to nitrocellulose, and probed with HRP-neutravidin(Pierce, Rockford, Ill.). The chemiluminescent signal was measured usingan EPI-CHEM II™ darkroom system (UVP Bioimaging Systems, Upland,Calif.). Concentrations of eluted b-βγ dimers were determined bycomparing to a standard curve of fully purified 100% biotinylated Gβ1γ2from at least two separate gels.

(2) b-βγ Binding Assay

Phage ELISA assays used to assess peptide binding to wild-type andmutant b-βγ were performed according to standard methods (Smrcka andScott (2002) Methods Enzymol. 344:557-76). Briefly, 1 μg streptavidinwas immobilized in the well of a 96-six well plate overnight at 4° C.The wells were blocked with 100 μL of 2% bovine serum albumin (BSA) inTris-buffered saline (TBS) for 1 hour at 4° C. followed by three washesof IX TBS/0.5% TWEEN®. Forty μL of 25 nM bGβ1γ₂ in TBS/0.5% TWEEN® wasadded to each well and incubated at 4° C. for 1.5 hour. The wells werewashed, followed by the addition of IxIO⁶ to IxIO¹⁰ phage particles andincubated at 4° C. for 3 hours. The wells were then washed six timeswith TBS/0.5% TWEEN® followed by addition of 40 μL of 1:5000 dilution ofanti-M13 antibody (Pharmacia, Uppsala, Sweden) and incubated at roomtemperature for 1 hour. The wells were washed, followed by the additionof 40 μL of (2,2′-Azino-bis (3-ethylbenzthiazoline)-6-sulfonic acid(ABTS) and the colorimetric reaction was monitored at 405 nm.Nonspecific binding was subtracted for each reading.

Signals obtained with partially purified b-βγ subunits were similar tosignals obtained from fully purified b-βγ subunits. Blocking ofGαi^(>>)Gβ1γ₂ binding was assessed by simultaneously adding 200 pMFITC-Gαi with or without SIGK to 50 pM immobilized b-βγ and measuringthe amount of FITC-Gαi bound to the beads by flow cytometry according tostandard methods (Ghosh, et al. (2003) supra; Sarvazyan, et al. (1998)J. Biol. Chem. 273:7934-40).

b) Results

(1) Architecture of the Gβ1γ₂<<SIGK Complex

Unless indicated otherwise, amino acid residues having the prefix “s”are indicative of SIGK residues.

Gβ1 is a β-propeller composed of seven four-stranded β-sheets (“blades”)and an N-terminal extended helix that interacts extensively with Gγ₂.Each sheet is composed of WD-40 repeats connected by loops of variablelength. Residues 2-340 of Gβ1 are included in the model. B factorsthroughout the core of Gβ1 are less than 40 A². Residues with Bfactors>60 A² are found in three loop regions: Lys127-Ser136 in bladetwo, Arg214-Met217 in blade four, and Ser265-Ile269 in the loopconnecting blades six and seven. Gγ₂ forms a helix with a kink made byresidues Asn24-Lys29 and a coil region beginning at residue His44. Theaverage B factor within the Gy₂ molecule is 44 A². No electron densitywas observed for the N-terminal seven residues and the C-terminalsixteen residues of Gγ₂ or the prenyl lipid modification at theC-terminus of Gγ₂.

SIGK forms an α-helical structure broken by a glycine at position 10.The C-terminal three residues form an extended structure that stretchesaway from the Gp₁ molecule and is supported by crystal contacts betweensPro12 and sAsp13 with Thr47 and Lys337 from a symmetry-related Gβ1molecule. The B factors for the N- (sSer1, slle2) and C-terminal(sGly1O-sAsp13) residues of SIGK are greater than 50 A²; those for allother residues are between 30-50 A². The electron density for the mainchain atoms in residues 1-13 is well-defined; three of the SIGK sidechains that do not contact Gβ1 (slle2, sLys7, and sAsp13) aredisordered. The peptide binds across the “top” face of Gβ1 and is buried970 A² total solvent-accessible surface area. The peptide makes nocontact with the Gγ₂ subunit, which is bound to the “bottom” surface ofthe Gβ1 torus.

The SIGK contact surface on Gβ1 was separated into two regions: anacidic region on Gβ1 that interacts with the N-terminus of the peptide,and a largely nonpolar region that interacts with the C-terminus of thepeptide. In total, thirteen Gβ1 residues directly contact SIGK,contributed by six of the seven blades of the β-propeller (Table 11).

TABLE 11 Gβ₁-Interacting SIGK-Interacting Distance Type of ResiduesResidues ({acute over (Å)}) Interaction Lys57 Cε Leu9 O 3.35 Nonpolar CεGly10 Cα 3.99 Nonpolar Tyr59 OH Leu9 O 2.66 Polar Cε Ile8 O 3.87Nonpolar Trp99 Nε1 Tyr11 OH 2.81 Polar Cδ1 Leu9 Cδ2 3.59 Nonpolar Val100O Leu9 Cδ2 3.75 Nonpolar Met101 Cε Ile8 Cγ2 3.46 Nonpolar Cε Ala5 O 3.52Nonpolar Cε Leu9 Cδ2 3.54 Nonpolar Leu117 Cδ1 Ile2 Cy2 3.46 Nonpolar Cδ2Ala5 Cβ 3.68 Nonpolar Cδ2 Leu9 Cδ1 3.80 Nonpolar Tyr145 Cε2 Ser1 O 3.19Nonpolar OH Lys4 Cγ 3.45 Nonpolar Cδ2 Ala5 Cβ 3.81 Nonpolar Asp186 Oδ2Ser1 O 3.03 Polar Met188 Cε Ile8 Cδ1 3.31 Nonpolar Cε Lys4 Cε 3.48Nonpolar Asp228 Oδ2 Lys4 Nζ 3.23 Polar Asn230 Nδ2 Lys4 Nζ 2.82 PolarAsp246 Oδ2 Lys4 Nζ 3.05 Polar Trp332 Cζ2 Ile8 O 3.12 Nonpolar CH2 Gly10Cα 3.57 Nonpolar

The N-terminal binding surface is centered on an electrostaticinteraction in which sLys4 projects into a negatively charged bindingpocket on Gβ1γ₂ where it forms hydrogen-bonded or charge interactionswith Asp228, Asn230, and Asp246. A hydrogen bond between the carbonyloxygen of Asp228 and the main chain nitrogen of Asp246 stabilizes thethree acidic residues on Gβ1. Met188 participates in van der Waalsinteractions with the alkyl chain of sLys4, and Asp186 forms a polarcontact with the carbonyl oxygen of sSer1 and also makes a hydrogen bondto the amide of Cys204. Additionally, Tyr145 forms van der Waalsinteractions with the main chain oxygen of sSer1, the sLys4 side chain,and the Cβ atom of sAlaδ, and forms a hydrogen bond with the nearbyamide of Gly162. The side chain of Leu117 is within van der Waalscontact distances of the side chains of slle2 and sAla5. Together, thesenine Gβ1 residues form a surface that tethers SIGK to Gβ1 using chargedand nonpolar interactions. Mutational analysis of SIRK and SIGK peptidescan now be interpreted in the context of the SIGK*Gβ1γ₂ structure(Scott, et al. (2001) supra; Goubaeva, et al. (2003) supra). Wild-typeSIRK peptide inhibits the activation of PLC β2 by Gβ1γ₂ with an IC₅₀ of5 μM. Substitution of sLys4 with alanine in the SIRK peptide lowers theIC₅₀ of the peptide 12-fold, and mutation of sAlaδ to glycine lowers theIC₅₀ by 13-fold. Mutation of slle2 to alanine reduces IC₅₀ of thepeptide by 4-fold, and mutation of sSer1 to alanine has no effect onIC₅₀ (Scott, et al. (2001) supra). The SIGK^(>>)Gβ1γ₂ structureindicates that the main chain of sSer1 and the side chains of slle2,sLys4, and sAla5 contact multiple resides on Gβ, thereby explaining thismutational data.

To measure the contribution of the Gβ1 residues observed at theGβ1γ₂>>SIGK interface to the binding energy for the complex, twoapproaches were utilized. First, an ELISA assay was used to measurebinding of immobilized Gβ1γ₂ subunits to phage displaying the SIGKsequence (Table 12). The ELISA binding data were then correlated withIC₅₀ values for SIGK as a competitor of Gβχγ₂ association with Gαu(Table 13). Both assays were then carried out with Gβ1γ₂ heterodimerscontaining mutations in the Gβ₁ subunit. In the N-terminal bindingsurface, mutation of Gβ₁ Asn230 to alanine decreased the affinity ofGβ₁γ₂ for peptide 10-fold (Table 12). Single mutation of Gβ1 residuesAsp186, Met188, Tyr145, and Leu117 to alanine also resulted in Gβχγ₂dimers with drastically decreased affinity for SIGK (Table 12).Gβ1mutants in which either Asp228 or Asp246 were substituted withalanine did not dimerize with Gγ₂ and therefore were not analyzed.However, a mutant in which Asp228 was substituted with serine causedonly a slight loss in binding affinity for SIGK peptide (Table 12).Thus, many of the Gβ1 residues that create the N-terminal SIGK bindinginterface contribute strongly to the energy of binding.

TABLE 12 % of Wild-Type Signal Gβ₁γ₂ Mutation (mean ± SD) Lys57Ala 18.6± 4.6  Tyr59Ala 24.7 ± 15.2 His62Ala 111.2 ± 11.3  Trp99Ala 66.0 ± 7.7 Met101Ala 32.2 ± 15.5 Leu117Ala 2.1 ± 2.4 Tyr145Ala 0.8 ± 0.9 Asp186Ala13.0 ± 13.1 Met188Ala 2.5 ± 3.7 Asn230Ala 22.4 ± 4.2  Asp246Ser 66.5 ±7.5  Phe292Ala 109.1 ± 21.4  His311Ala 94.3 ± 18.9 Arg314Ala 50.2 ± 5.0 Trp332Ala 7.1 ± 3.7 Amino acids that contact the SIGK peptide wereindividually mutated to alanine (or serine for Asp246) and binding topeptide was assayed using a phage ELISA. Immobilized b-βγ was incubatedwith phage displaying SIGK peptide. Phage binding was detected using anα-phage antibody; the raw data was absorbance at 405 nm. Data shown arethe mean ± SD of triplicate determinations from three independentexperiments.

TABLE 13 Log % Maximal Fα Binding (±SD) [SIGK] M Wild-Type Met188AlaTrp332Ala Arg314Ala −7 100.0 ± 0.0  100.0 ± 0.0  100.0 ± 0.0  100.0 ±0.0  −5.7 55.0 ± 10.8 101.3 ± 7.6  72.7 ± 2.3 65.3 ± 5.8 −5 45.7 ± 16.380.7 ± 7.6 57.3 ± 7.2  47.0 ± 11.1 −4.7 17.3 ± 3.8   72.7 ± 11.2 33.0 ±3.5 30.7 ± 4.2 −4.4 13.3 ± 1.5  40.0 ± 7.1 24.7 ± 3.5 21.0 ± 0.0 −4.15.8 ± 4.8 33.0 ± 7.2 16.7 ± 1.2 13.0 ± 3.0 SIGK competition forFITC-Gαiiβiγ₂ interactions with representative Gp₁ subunit mutants. SIGKand FITC-αn were simultaneously added to streptavidin beads coated withwild-type or mutant b-βγ protein and the amount of FITC-αn bound to thebeads was assayed by flow cytometry. Data are shown as the mean oftriplicate determinants +/− standard deviation of a representativeexperiment. The experiment was repeated two (Met188A) or three(wild-type, Arg314A, Trp332A) times with similar results. Comparison ofthe two assays over a selection of mutants that spanned the range ofSIGK binding affinities indicates that a 50% loss of binding translatesinto a five-fold increase in IC₅₀, a 75% loss of binding corresponds toa 10-fold increase, a 90% loss is a 20-fold shift and a 98% loss is a50-fold shift. The IC₅₀ values are as follows: wild-type = 0.47 μM,Arg314A = 1.5 μM, Trp332A = 9 μM, and Met188A = 22 μM.

The second area of binding involves most of the C-terminal residues ofSIGK (sAla5-sGly11), which pack against a largely hydrophobic pocket onGβ1. This pocket extends HA from Trp332 on blade seven to Met188 inblade two. Eight Gβ1 residues are in direct contact with the C-terminalsurface of SIGK, and two more Gβ1 residues support the residues directlyinvolved in the SIGK interaction. Met188, which interacts with sLys4 inthe N-terminal interface, is also within contact distance of the sidechain of sLeu8. SIGK residues sAla5, sLeuδ and sLeu9 are complimented byvan der Waals interactions with Leu117, Met101, Trp99, Tyr59 and thealkyl chain of Lys57. The main chain oxygen of Val100 interacts with theside chain of sLeu9. The indole imine of Trp99 forms a hydrogen bondwith the hydroxyl group of sTyr11 and the side chain of Trp332 makescontact with the main chain oxygen of slle8 and the Ca of sGly1O. Theside chains of Lys57 and Arg314 are positioned on either side of Trp332and support its orientation in the binding site. Arg314 also forms ahydrogen bond with Trp332, and Lys57 with the nitrogen of Gln75, furtherstabilizing this interaction surface on Gβ1. Data from alanine scanningof the peptide (Scott, et al. (2001) supra; Goubaeva, et al. (2003)supra) validate these structural observations. Mutation of slleδ, sLeu9or sGly1O to alanine increases the IC₅₀ for inhibition of PLC activationby 40-fold (5 μM to 200 μM), 60-fold and 12-fold, respectively (Scott,et al. (2001) supra). The same mutation of sLeu9 also blocks the abilityof SIRK to enhance ERK1/2 phosphorylation in RASM cells (Goubaeva, etal. (2003) supra). Mutation of amino acids in Gβ1 that constitute theSIGK C-terminal binding surface caused a loss in affinity for the SIGKpeptide, although to different extents. Mutation of Leu117, Met188, orTrp332 to alanine nearly abrogated SIRK binding; mutants of Lys57,Tyr59, Met101, and Arg314 had more modest effects (Table 12 and Table14). The Trp99 mutation resulted in a 4-fold decrease in affinity. Asummary of all the Gβ1 mutations (i.e., conversions to alanine)presented herein and their effects on SIGK binding affinity is listed inTable 14.

TABLE 14 Loss in Affinity for SIGK Peptide 75-100% 50-75% 25-50% 0-25%No Effect Gβ₁ Residue Lys57 Met101 Trp99 His311 His62 Tyr59 Arg314Asn246 Phe292 Leu117 Tyr145 Asp186 Met188 Asn230 Trp332

Considering all of the data for the N-terminal and C-terminal SIGKbinding interfaces, seven of the fifteen residues of the SIGK peptideand ten of the twelve Gβ residues tested contribute significant bindingenergy to the interface, in good correlation with the structural model.

The binding surface of Gβ1 in the Gβ1γ₂>>SIGK complex is notsignificantly changed upon SIGK binding. The RMSD between the coreresidues of Gβ1 in the Gβ1γ₂<<SIGK complex and that in the uncomplexedGβ1γ_(x) heterodimer (ITBG (Sondek, et al. (1996) Nature 379:369-74);Va140-Asn340, Ca only) is 0.88 A. However, the side chains of Trp99,Tyr59, Asp228, Leu117 and Met101 rotate to accommodate SIGK such thatatoms within these residues undergo maximum displacements of 4.0 A, 3.6A, 2.9 A, 2.8 A and 2.3 A, respectively, relative to their positions inuncomplexed Gβ1. The B factors for residues in the SIGK binding surfaceare close to the overall average for the complex. However, the B factorfor Trp99 is reduced two-fold upon binding to SIGK, as indicated bycomparison of normalized B factors of the respective structures. In thisanalysis, there are no large conformational changes or disorder to ordertransitions in Gβ upon SIGK binding. The SIGK^(>>)Gβ1γ₂ complex may becompared to those of five Gβ1γ₂ complexes with protein targets: theGβ1γ₂′Gαii heterotrimer (1GG2) (Wall, et al. (1995) supra; Wall, et al.(1998) supra) and the Gβ1Yi′Gαt/1 heterotrimer (IGOT) (Lambright, et al.(1996) supra), the Gβ1γi<<phosducin complex (IAOR and 2TRC) (Loew, etal. (1998) supra; Gaudet, et al. (1996) supra), and the Gβ1γ₂>>GRK2complex (10 MW) (Lodowski, et al. (2003) supra). Superposition of theGβ1Y₂#SIGK complex with each of these structures yields average RMSdeviations for Gβ1 residues 40-340 of less than 1.0 A (Ca only). Withthe exception of a few residues involved in the Gβ1Yi′phosducin complex,the Gβy heterodimer does not undergo significant structuralrearrangement in order to bind protein targets, nor does it in theGβ₁γ₂<<SIGK structure.

(2) Measurement of α-βγ Interactions via Flow Cytometry

Fluorescein-labeled Ga^ (Fan) was prepared in accordance with standardmethods (Sarvazyan, et al. (1998) supra). Assays were used to determinepeptide effects on Gα-Gβγ interactions included competition anddissociation assays (Ghosh, et al. (2003) supra). Briefly, forcompetition-based assays, 100 pM Fan and indicated concentrations ofpeptides were added to 50 pM b-Gβ1γ₂ immobilized on 10^(s) beads per mLand incubated at room temperature for 30 minutes to reach equilibrium.The bead-associated fluorescence was then recorded in a BD BiosciencesFACSCALXBUR™ flow cytometer. Data was corrected for backgroundfluorescence and fit with a sigmoid dose response curve using Graph PadPrism 4. To measure dissociation of Fan from b-Gβaγ₂, 100 pM of Fa^ wasincubated with 50 pM immobilized b-Gβ1γ₂ at room temperature for 15-20minutes. The fluorescence of bound Fαii subunit was measured, followedby the addition of a 200-fold excess of unlabeled Gcx±_(x) or peptidesand the amount of Fan remaining bound to the beads was measured at theindicated times.

(3) Molecular Recognition at the Protein Interaction Site

Having demonstrated that the interface for SIGK peptide binding wasdivided into two broad interactions; a C-terminal binding interface,which contacts the hydrophobic core of the peptide (amino acids 8-10,Ile-Leu-GIy), and an N-terminal interface, which associates with theN-terminus (Lys4 primarily) of the peptide, the molecular basis forrecognition of the peptide was determined. Accordingly, amino acids ofthe common binding surface of Gβ1 were individually alanine substitutedto determine which amino acids were most critical for the interaction ofGβ1γ₂ with nine different SIGK peptide derivatives (Table 15).

TABLE 15 Phage Name Sequence* SEQ ID NO: Group 3.14 SIGKALFILGYPDYD 5 I2F LCSKAYLLLGQTC 6 C1 SCKRTKAQILLAPCT 7 C14 WCPPKAMTQLGIKAC 8 II 3CSCGHGLKVQSTIGACA 9 C4 SCEKRYGIEFCT 10 III C5 SCEKRLGVRSCT 11 C8SCARFFGTPGCT 12 C2 WCPPKLEQWYDGCA 13 IV *Underlined residues denote thelysine residue contacting the N-terminus, and the hydrophobic coreresidues.

The nine peptides were selected to represent the different consensusgroups of peptides previously identified (See Scott et al. (2001) supra;Table 15) and to compare binding characteristics within and betweenconsensus groups. Binding of phage displaying these peptides towild-type Gβ1γ₂ gave ELISA signals that were different, but fell withina similar range (25 to 100% binding relative to phage 3.14). Asdisclosed herein, the binding signal obtained in the ELISA assay wascorrelated to a loss in affinity by comparing the results to behavior ofthe peptide in a solution based assay. For example, a mutant displayingan 80% loss of binding in an ELISA had a corresponding 10-fold shift inpeptide affinity in solution. For the purposes of present disclosure,any substitution that decreased the binding to less than 20% of thewild-type binding was considered to be a critical binding contact forthat peptide. Data obtained from this analysis is presented in Table 16.

TABLE 16 % of Wild-Type Signal C-Terminal Interface Shared PeptideTrp332 Lys57 Tyr59 Trp99 Leu117 Met101 Met188 Group I 3.14 7.1 ± 3.718.6 ± 4.6  24.7 ± 15.2 66.0 ± 7.7 2.1 ± 2.4 26.8 ± 12.5 2.5 ± 3.7 2F−1.5 ± 3.6  −4.5 ± 6.2  −2.0 ± 9.5   32.6 ± 26.3 2.1 ± 5.5 6.0 ± 5.1 8.5± 5.2 C1 −0.3 ± 2.5  0.1 ± 1.6 0.3 ± 0.7  1.2 ± 2.7 5.2 ± 3.1 70.5 ±35.3 4.0 ± 3.4 Group II C14 1.6 ± 2.4 3.0 ± 5.0 3.6 ± 2.0 10.1 ± 3.3 3.8± 9.2 1.1 ± 4.9 9.9 ± 3.8 3C −0.3 ± 1.7  3.6 ± 7.5 8.5 ± 6.4 −0.5 ± 5.9  10 ± 13.3 −4.2 ± 5.5  −5.7 ± 5.6  Group III C4 1.7 ± 3.0 −0.2 ± 1.9 7.6 ± 9.6  67.0 ± 15.2 18.4 ± 7.1  61.2 ± 30.9 127.5 ± 22.4  C5 3.2 ±3.7 5.6 ± 5.3 73.8 ± 16.6 39.0 ± 3.8 28.5 ± 5.2  47.8 ± 18.9 97.2 ± 14.2C8 0.7 ± 2.4 14.2 ± 9.8  4.0 ± 6.2 −0.8 ± 2.7 24.7 ± 12.5 23.6 ± 8.5 122.6 ± 26.2  Group IV C2 4.7 ± 6.2 −1.2 ± 4.5  −1.7 ± 4.8  −1.0 ± 5.11.5 ± 5.5 157.1 ± 51.5  −0.7 ± 2.5  % of Wild-Type Signal N-TerminalInterface Indirect Peptide Asn230 Asp246 Tyr145 Asp186 His311 Arg314Group I 3.14 22.4 ± 4.2  66.5 ± 7.5  0.8 ± 0.9 14.4 ± 13.4 93.4 ± 21.550.2 ± 5.0  2F 27.6 ± 19.1 0.6 ± 2.5 0.1 ± 6.5 2.4 ± 3.5 23.6 ± 46.0 2.4± 3.9 C1 19.8 ± 12.3 2.3 ± 3.0 88.0 ± 29.9 1.4 ± 1.7 3.0 ± 4.0 2.8 ± 1.6Group II C14 60.0 ± 26.5  6.3 ± 13.9 1.9 ± 5.9 3.6 ± 1.9 6.1 ± 3.4 3.9 ±7.9 3C 4.1 ± 4.4 2.0 ± 2.7  2.7 ± 10.5 35.0 ± 17.2 30.5 ± 18.9 8.3 ± 3.6Group III C4 11.5 ± 6.0  35.8 ± 7.4  4.4 ± 3.8 51.3 ± 15.0 36.5 ± 8.3 1.4 ± 1.0 C5 33.5 ± 7.2  56.3 ± 4.1  16.7 ± 4.8  45.7 ± 5.2  58.7 ± 14.476.5 ± 6.7  C8 74.8 ± 14.8 60.8 ± 14.4 17.5 ± 7.8  124.9 ± 29.1  51.6 ±13.2 20.8 ± 11.1 Group IV C2 0.0 ± 3.3 5.9 ± 5.7 4.5 ± 8.1 267.2 ± 40.6 11.7 ± 8.0  1.3 ± 3.1 Wild-type or alanine-substituted biotinylatedGβχγ₂ subunits were immobilized on a streptavidin-coated 96-well plate,followed by the addition of phage. Phage binding was assessed asdescribed herein. Data was corrected for nonspecific binding of phage tothe plate and is represented as a percent wild-type binding. Data shownare mean ± SD of duplicate determinations from three independentexperiments.

Unexpectedly, each of the peptides utilized unique combinations of aminoacids within the SIGK binding surface for its particular interaction. Adominant feature amongst the peptides was a strong requirement forTrp332, within the C-terminal interface. Lys57, Tyr59, Leu117, alsowithin this interface, generally contributed significantly to bindingthe peptides, though there were cases where their effects were notabsolutely required. The remainder of the amino acids had more variableeffects on binding of each peptide. For example, SIGK has a minimalrequirement for Trp99 whileSer˜Cys-Lys-Arg˜Thr-Lys-Ala-Gln-Ile-Leu-Leu-Ala-Pro-Cys-Thr (Cl; SEQ IDNO: 7) absolutely requires Trp99 for binding. The reverse is true forTyr145 where SIGK binding has an absolute requirement for Tyr145 andSer-Cys-Lys-Arg-Thr-Lys-Ala-Gin-He-Leu-Leu-Ala-Pro-Cys-Thr (Cl; SEQ IDNO: 7) binding is not affected by this mutation.

The N-terminus of SIGK interacts with the Gβ subunit through two maincontacts: sSer1 interactions with βAsp188 and βTyr145 residues, andsLys4 interactions with βMet188 through a Van der Waals interaction andβAsn230, βAsp246 and βAsp228 through hydrogen bonded or chargedinteractions. In the expression system utilized herein, Asp228Ala andAsp246Ala did not dimerize with gamma and could not be purified;however, Asp246Ser was expressed and purified. In general, peptides ingroups I, II and IV have a substantial requirement for binding to theN-terminal region, reflected by an almost complete loss of binding tothe Met188Ala and Asp246Ser (except SIGK) mutants and variousrequirements for Asn230.

Peptides in groups I, II and IV have a conserved motif where a lysine isspaced three amino acids away from a hydrophobic core motif (see Table15). This motif in SIGK provides the appropriate spacing in a singlealpha-helical turn between the lysine that interacts with the N-terminalbinding surface and the Ile-Leu-Gly motif that interacts with theC-terminus. It is believed that some of the other peptides adopt asimilar α-helical structure that may make this spacing critical. Thepeptides in group III bind the C-terminal interaction region, but lack arequirement for Met188 and have minimal requirements for Asn230 andAsp246, indicating they do not use the N-terminal binding surface fortheir interaction with β.

Two amino acids that do not apparently bind directly to SIGK were alsoanalyzed, Arg314 and His311. Replacement of Arg314 results in a modestdecrease in SIGK binding; however, for other peptides, Arg314 isabsolutely required indicating that they may directly interact with thisamino acid. His311 lies well outside the SIGK peptide binding site butwas mutated because of its potential involvement in a conformationchange in βγ subunits (Gaudet, et al. (1996) supra; Loew, et al. (1998)supra). The imidazole side chain of His311 is 13 A from the guanidonitrogen of Arg314, the closest amino acid that apparently interactswith any of the peptides. It is unlikely that His311 could directlyinteract with amino acids from the phage display-derived peptides.Nevertheless, mutation of His311 to alanine affected binding of variouspeptides to varying extents. Peptides whose binding was affected byHis311A also required Arg314 for binding, an effect possibly due to analteration in the position of Arg314.

It has been demonstrated that two peptides predicted to bind at theGα-Gβγ interface, βARK-ct peptide (amino acids 643-670) and QEHA,blocked heterotrimer formation but could not promote heterotrimerdissociation (Ghosh, et al. (2003) supra). The crystal structure of theGRK2 (βARK)-Gβγ complex reveals that the surface interacting with theβARK-ct peptide partially overlaps with the SIGK and Gα-switch IIbinding site (Lambright, et al. (1996) supra; Wall, et al. (1995) supra;Lodowski, et al. (2003) supra). In particular, amino acids Trp99,Trp332, and Try59 within the hydrophobic pocket are common interactionsites in all three structures. The SIGK peptide and α switch II have alysine residue occupying nearly identical positions on Gβ. Although theβARK-ct peptide has a lysine residue in a similar position, the geometryand nature of the interaction is different. βARK interacts only withAsp228 whereas SIGK and Ga interact with Asp228, Asp246, Asn230 andMet188. Based on this difference, it was determined whether the specificinteractions of SIGK at this interface were critical for promotingdissociation.

To examine subunit dissociation, the SCAR peptide, another peptidederived from the phage display screen, was used. Amino acids within theN-terminal interaction interface, Asn230, Asp246 and Met188, contactingsLys4 of SIGK, are not important for binding SCAR. SCAR lacks a lysineresidue with the correct positioning relative to the hydrophobic coremotif to reach the lysine-binding N-terminal surface (Table 15).Therefore, SCAR would not be able to promote subunit dissociation. BothSIGK and SCAR can compete with Ga± for binding to Gβχγ₂, with IC₅₀'s of0.5 and 1.7 μM, respectively. However, unlike the SIGK peptide,saturating concentrations of SCAR peptide could not promote dissociationof a preformed heterotrimer. Concentrations of up to 160 μM SCAR, (fourtimes the saturating concentration) did not cause dissociation. Theinability of SCAR to promote heterotrimer dissociation was not due toits lower binding affinity since SIRK has a similar affinity andpromotes dissociation. These results indicate that peptide binding tothe N-terminal interface is necessary for acceleration of heterotrimerdissociation.

To more directly assess the importance of peptide binding to theN-terminal peptide binding interface, the sLys4 residue of SIRK wasmutated to alanine, eliminating the key contact to the N-terminalbinding pocket. This peptide had a markedly lower affinity than SIRK(IC₅₀=60 μM vs 1.4 μM) for blocking Gα-Gβγ interactions; however, athigh concentrations, it blocked to levels near that of SIRK. Despiteblocking Gα-Gβγ interactions, SIRK (Lys4Ala) failed to accelerateheterotrimer dissociation. The apparent off-rate of Fα{umlaut over (υ)}appears slower for SIRK(LyS4Ala) relative to the intrinsic dissociationrate. This could be because SIRK(Lys4Ala) is low affinity blocker, andis not effective at preventing rebinding of Fan. To confirm that the lowaffinity of SIRK(Lys4Ala) was not responsible for the inability toaccelerate dissociation, a peptide with comparable affinity to SIRK(Lys4Ala), SIRK(GIyIOAIa) (IC₅₀˜80 μM), was tested. This peptide hasLys4 but Ala is substituted for GIy at position 10, thus SIRK(GIyIOAIa)retains binding to the N-terminal interface but has a reduced affinitydue to decreased interactions with the C-terminal region. SIRK(GIyIOAIa)blocked heterotrimer formation at high peptide concentrations anddespite having a low affinity for Gβγ, could still accelerateheterotrimer dissociation. SIGK binds to Gβ_(α) at a region occupied bythe switch II domain of Ga subunits in the heterotrimer. The crystalstructure of the heterotrimer reveals the switch interface (composed ofswitch I and switch II) of Ga buries approximately 1,800 A of Gβ throughnumerous contacts (Lambright, et al. (1996) supra; Wall, et al. (1995)supra) ; however, the effects of mutations of β subunit amino acids atthis interface on α subunit binding have not been measured in directbinding assays near the K_(d) for Gα-Gβγ interactions. Switch I andswitch II undergo large conformational changes upon GTP binding and itis thought these changes mediate heterotrimer dissociation.

Gβ1 subunit mutants disclosed herein were isolated from insect cells asa complex with Gy₂ and hexa-histidine-tagged Gαii indicating that manyof these contacts between the subunits predicted from the crystalstructures were not individually critical for Ga subunit binding. Todetermine which amino acids were contributing to the ability of peptidesto enhance dissociation rate constants, the dissociation rate constant(k_(O)f_(f)) for Fαii from each of the individually substituted b-βiγ₂mutants was measured. The intrinsic off-rate for wild-type was 0.123s-1, corresponding well with previous measurements (Sarvazyan, et al.(1998) J. Biol. Chem. 273:7934-7940). Data from all of these mutants areshown in Table 17.

TABLE 17 Mutation K_(off)** Wild-Type 0.123 ± 0.0429 min⁻¹ Lys57Ala0.144 ± 0.0441 min⁻¹ Tyr59Ala 0.181 ± 0.0726 min⁻¹ Trp99Ala 0.288 ±0.0547 min⁻¹ Met101Ala 0.114 ± 0.0175 min⁻¹ Leu117Ala 0.361 ± 0.0258min⁻¹ Tyr145Ala 0.155 ± 0.0423 min⁻¹ Asp186Ala 0.160 ± 0.0429 min⁻¹Met188Ala 0.122 ± 0.0380 min⁻¹ Asn230Ala 0.148 ± 0.0488 min⁻¹Asp246Ser^(†) — Arg314Ala 0.118 ± 0.0246 min⁻¹ Trp332Ala 0.301 ± 0.0420min⁻¹ **Mean + SD from four independent experiments. ^(#)Statisticallysignificant as compared to wild-type (p < 0.05) as determined by aone-way ANOVA followed by independent linear contrasts. ^(†)k_(Off)could not be measured because significant stable binding of F-αi was notdetectable.

The results showed that of the 12 mutants tested, Trp99Ala, Leu117Ala,and Trp332Ala were statistically different from wild-type withrelatively minor increases in k_(off). On the other hand, Asp246Ser,despite being able to be purified based on 6HisGαi binding (although inlow yield from a large culture), was unable to stably bindF-α_({umlaut over (υ)}) in the flow cytometry assay at the lowconcentrations used for this assay. This indicates that interactionswith Asp246 are critical for stable Ga subunit interactions, whileindividual interactions in the primarily hydrophobic C-terminalinterface are not as important.

(4) Small Molecule Library Screen

A phage ELISA assay was used to determine whether small moleculesidentified in the computational screen could interact with the Gβyprotein interaction surface. Phage displaying the SIGK peptide were usedin accordance with established methods (Scott, et al. (2001) supra;Smrcka and Scott (2002) supra). The screen was based on a reduction inthe optical density (OD) of wells containing Gβγ subunits and phage. Ineach plate, three wells contained positive controls for binding thatincluded b-βγ subunits, SIGK-phage, and the appropriate amount ofvehicle. Three background wells contained no βy subunits.

As disclosed herein, biotinylated Gβy subunits were immobilized on thesurface of a 96-well plate coated with streptavidin, phage displayingGβγ-binding peptides were subsequently added and binding in the presenceand absence of test compounds detected with an anti-phage antibody.

(5) Inhibition of Gβγ Signaling in Neutrophils

Ca²⁺ fluxes were measured using two 35 mL cultures of differentiatedHL-60 neutrophil cultures (0.2×10⁶ cells/mL). Cells were cultured forthree days with in DMSO

(1.2%), washed in HSS and resuspended in 2 mL HBSS at a concentration of7×10^(s) cells/mL. Addition of DMSO to the growth medium inducesdifferentiation of these cells into morphologically and functionallymature neutrophils

(Collins, et al. (1978) Proc. Natl. Acad. Sci. USA 75:2458; Collins, etal. (1979) J. Exp. Med. 149:969). Neutrophils were preloaded with fura-2(1 μM), a fluorescent Ca²⁺-sensitive indicator (Suh, et al. (1996) J.Biol. Chem. 271:32753), for 45 minutes, washed with HBSS and resuspendedin 2 mL of indicator-free HBSS. An 140 μL aliquote of cells was added toa total of 2 mL HBSS. Fluorescence ratios were taken by dual excitationat 340 and 380 nm and emission at 510 nm. After a stable baseline wasestablished, either DMSO or NSC119910 was added and incubated for 5minutes. Subsequently, either fMLP or ATP agonists were added toactivate release of Ca²⁺ from intracellular stores.

2. Example 2 Pharmaceutical Manipulation of G Protein βγ SubunitSignaling

a) Analysis of Selectivity Profiles of βγ-Binding Compounds.

The specificity of compounds are further characterized in vitro withrespect to effector functions by analyzing effects on βγ-dependentregulation of adenylyl cyclase I and II, N-type Ca²⁺ channels andinwardly rectifying K⁺ channels. Since βγ subunits are criticalcomponents of receptor-G protein coupling, compound effects on thereceptor stimulated GTP binding and turnover are evaluated, anddetermined if compound binding kinetics affects the ability to influencereceptor G protein coupling. Selectivity of compounds for G protein βγsubunit subtypes is also evaluated. Medium throughput assays aredeveloped that are used to predict effector selectivity and potentiallytherapeutic efficacy of new compounds without laborious screening inenzymatic assays. These experiments form the basis for connectingspecificity characteristics with biophysical binding characteristics.

b) Determination of the Molecular Basis for Binding and Selectivity ofGβγ-Binding Compounds.

Small molecules binding directly to G protein βγ subunits selectivelyalter individual protein-protein interactions by binding to differentsubsites of the G protein βγ subunit “hot spot”, but compound chemistryand/or binding kinetics impart some degree of selectivity. Compoundspredicted to bind to the same site but with different chemistries aretested for effector selectivity in vitro. Surface plasmon resonance isused to measure binding kinetics and dissociation constants forindividual selective compounds coupled with site-directed mutagenesis tounderstand binding characteristics and define binding sites forindividual selective compounds. Specific compounds are co-crystallizedwith Gβγ to give a detailed picture of the interactions betweenselective compounds and Gβγ.

c) Computational Prediction of βγ Binding Specificity and Screening ofNew Libraries.

A specific binding subsite for a specific selective compound, defined byeither X-ray structure determination or mutagenesis, is used as thespecific target in a computation screen of a 50,000 compound diversitylibrary. The top 100 compounds are analyzed in each of 7 scoringfunctions with the hypotheses that: 1) a single scoring function willemerge as the best scoring function for that site which will simplifyfurther screening. 2) Compounds predicted to bind to that site haveselectivity for βγ-effector interactions that resemble other compoundsthat bind to that subsite.

d) Analysis of Specificity and Efficacy in Neutrophil Based Models ofInflammation.

Specific selective compounds are analyzed for selective functionaleffects in neutrophils as a model for inflammation. A particular focusis on compounds that modify the βγ-dependent regulation ofphosphoinsotide 3 kinase γ (PI3Kγ). Specifically, compounds are testedfor specific effects on regulation of fMLP or IL-8 dependent signalingpathways in an HL60 neutrophil model cell line and in primary humanneutrophils. Compounds that are selective for effects on neutrophilsignaling are assayed for selective effects on neutrophil chemotaxis,superoxide production and extracellular matrix adhesion.

e) Background

(1) Interactions Between βγ Subunits, α Subunits, and Effectors.

(a) Structure of the Heterotrimer:

Three dimensional crystal structures have been solved for G proteinsyielding information about the interaction interfaces between α and βγsubunits and between these subunits and their effectors (Lambright etal., 1996; Noel et al., 1993; Tesmer et al., 1997; Wall et al., 1995).The β subunit belongs to the WD-40 β propeller family of proteins. The Nterminus of the gamma subunit forms a coiled coil interaction with theN-terminus of β that extends away from the β propeller while the Cterminal portion of γ forms an α helix that packs against the β subunitat blade 5 of the propeller. The α subunit has extensive interactionswith a portion of the top of the β subunit and the amino terminal αhelix of the α subunit interacts with the side of βγ at blades 1 and 7.There are no interactions between the α subunit and the γ subunit inthese three dimensional models. βγ subunits have also been crystallizedin a complex with phosducin, a molecule that binds to βγ subunits in thevisual signal transduction system (Gaudet et al., 1996). Comparison ofthe phosducin structure with the heterotrimer structure reveals that thebinding site on βγ subunits for phosducin overlaps extensively, but notcompletely, with the binding site for α. This suggests thateffector-binding sites on βγ may only partially correspond to the αsubunit-binding site. On the other hand, in the recently solvedstructure of βγ subunits in a complex with G protein coupled receptorkinase 2 (GRK2), the main contacts of βγ with the pleckstrin homologydomain of GRK2 were very similar to the contacts with α subunits(Lodowski et al., 2003).

(b) Mutagenesis to Identify Protein Interaction Surfaces on βγ Subunits.

Different surfaces of βγ subunits are involved in interactions withdifferent effectors and target proteins. Evidence from a variety oflaboratories supports this view. To examine the functional role of the αsubunit binding interface of βγ subunits in effector activation, aseries of alanine mutants were made in the β subunit at amino acidsinvolved in contacts with the α subunit (Ford et al., 1998). Thesemutants were tested for activation of effectors, and it was found thatmany of the mutants were incapable of activating K⁺ channels,phospholipase C (PLC) β, and adenylyl cyclase (AC). Interestingly,distinct sets of amino acids at the α subunit interface seemed to beimportant for activation of different effectors. Putative effectorbinding sites have been identified in yeast β and γ subunits. Thesesites map to regions on β and γ subunits that do not correspond to the αsubunit-binding site (Leberer et al., 1992). A site for interaction ofPLC β2 with the amino terminus of β subunits that does not overlap withthe α subunit binding site was identified using chemical crosslinkingand mutagenesis (Yoshikawa et al., 2001). All of these experimentsindicate that proteins bind to βγ subunits with distinct but overlappingsets of interactions allowing for binding and regulation. Disclosedherein, peptides were identified that selectively block activation ofeffectors by βγ subunits in vitro, supporting this idea (Scott et al.,2001) and have more recently identified small molecule binders with evengreater specificity that are the subject of this proposal.

(2) Physiological Significance of βγ Activation.

G protein βγ subunit-mediated activation of effectors has diverse rolesin regulation of cell physiology. Some examples of cellular processesregulated by βγ subunits are briefly described here. In excitable cells,including neurons and cardiac myocytes, βγ subunits that are releasedfrom G_(i) regulate inwardly-rectifying K⁺ channels modulating membranepotential or heart rate (Clapham and Neer, 1997). In immune cells,chemokine receptors, such as the IL-8 receptor and the co-receptors forentry of the AIDS virus into leukocytes, are coupled to the release ofβγ subunits from G_(i) (Kuang et al., 1996; Littman, 1998). Severalmouse knockout studies implicate βγ regulated effectors in variousphysiological functions. For example, mouse neutrophils, withβγ-responsive PLCβ2 eliminated by gene-targeting, displayed increasedchemotaxis in response to chemotactic peptides, and the mice were moreresistant to viral infection (Jiang et al., 1997). In knockout micelacking βγ-regulated PLCβ3, morphine acting at G_(i) linked opioidreceptors produced painkilling effects at much lower doses (Xie et al.,1999). In a similar set of studies, genetic deletion of βγ-regulatedPI3Kγ resulted in decreased neutrophil migration and a reduction ininflammation.

Activation of multiple G_(i) and G_(q)-coupled receptors, includingthrombin, lysophosphatidic acid (LPA), and acetylcholine receptors,results in a mitogenic response in several cell types. MAP kinases arecritical components in the growth-promoting pathways regulated by thesereceptors. βγ subunits indirectly activate MAP kinase, indicating thatβγ subunits may mediate the growth-promoting effects of many Gprotein-coupled receptors (Gutkind, 2001; Luttrell et al., 1997).Sequestering βγ in smooth muscle cells inhibits serum stimulated growthand vascular restenosis (Iaccarino et al., 1999).

(3) G Protein βγ Subunits as a Target for Therapeutic Development.

The diverse functionality of Gβγ signaling in cellular physiologyindicated that manipulating G βγ function has significant therapeuticpotential. On the other hand Gβγ is known to be required for thefunctioning of all G protein coupled receptors so blocking all G βγfunctions might be predicted to have some side effects. The therapeuticusefulness of targeting Gβγ signaling has been investigated extensivelyusing the carboxy terminus of GRK2 (GRK2ct) (Bookout et al., 2003;Iaccarino et al., 1999; Iaccarino and Koch, 2003; Koch et al., 1995;Rockman et al., 1998) and to a lesser extent with other βγ bindingpeptides such as QEHA (Yao et al., 2002). GRK2ct, despite binding at theα/βγ “hot spot” interface interferes with βγ signaling to downstreamtargets without disrupting GPCR dependent G protein activation ingeneral. The basis for this selectivity is unclear. This has strongimplications for small molecule development, indicating that a strategythat targets the α/βγ interface “hot spot” is successful at blockingdownstream βγ signaling without disrupting G protein signaling ingeneral.

(4) βγ and Inflammation.

Data with knockouts of βγ effectors as described in “physiologicalsignificance of βγ activation” indicate that targeting βγ-effectorinteractions is a viable therapeutic strategy. Of particular interest isthe demonstration that deletion of PI3Kγ in mice inhibits neutrophilmigration in response to chemoattractants. PI3Kγ activity is directlyregulated by Gβγ released from chemokine and chemotactic peptidereceptors and is relatively selectively expressed in monocytic cells,indicating that blocking βγ-regulation of PI3Kγ is an effective strategyfor treating inflammatory diseases (Li et al., 2000). Because of theirroles in neutrophil recruitment, chemokines and chemokine receptors havebeen the subject of anti-inflammatory pharmaceutical development (Barneset al., 1998; Gong et al., 1997; Halloran et al., 1999; Ogata et al.,1997; Plater-Zyberk et al., 1997; Podolin et al., 2002; Yang et al.,2002). A potential problem is the overwhelming complexity of thesesignaling molecules (multiple chemokines, chemokine receptors, andredundancy) making it difficult to know which specific receptors totarget for conditions such as arthritis. Polychemokine (Carter, 2002) orcombinations of different chemokine (al Mughales et al., 1996)antagonists have been suggested, but there may be chemokines that act asan agonist at one receptor and an antagonist at another (Xanthou et al.,2003). An alternate approach that is currently being investigated isspecific pharmacological targeting of PI3K catalytic activity withinhibitors that are relatively selective for PI3Kγ relative to otherPI3K isoforms (Camps et al., 2005). In this approach, blocking PI3Kγcircumvents the problem with chemokine receptor redundancy by blocking acommon signaling target of chemokines. The small molecule inhibitorsthat were developed inhibit βγ-dependent activation of PI3Kγ indicatingan alternate approach to selectively blocking PI3Kγ relative to otherPI3K isoforms since these isoforms are not regulated primarily by Gβγ.

f) Results

Small molecules can be developed that by virtue of their small size,differentially affect activity of effectors that bind to the Gβγ “hotspot”. As discussed in Background and Significance, each effector has aunique footprint on the Gβγ surface with an overlapping binding surfaceat the βγ “hot spot”. Herein it is disclosed thateffector/pathway-selective molecules have been developed that appear tofunction by targeting the Gβγ “hot spot” in different ways. Thisrepresents a totally new approach to manipulation of G protein signalingbut the true mechanisms underlying selectivity remain to be defined.

(1) Amino Acid Sequence Characteristics of Peptides that AccelerateHeterotrimer Dissociation.

SIGK accelerates G protein subunit dissociation from a preformedheterotrimer leading to G protein activation, while other peptides thatinterfered with α/βγ interactions did not (Ghosh et al., 2003).Therefore, the specific interactions of SIGK at this interface werecritical determinants of the ability of a peptide to promotedissociation of Gβγ from Gα·GDP. To test this idea the ability of SCAR,which uses unique binding determinants for interactions with Gβγ, wasexamined to promote subunit dissociation. In an equilibrium bindingexperiment, both SIGK and SCAR peptide competed for binding offluorescently labeled α_(i1) (Fα_(i1)) to βγ in a flow cytometry basedbinding assay (FIG. 1B) (see (Ghosh et al., 2003; Sarvazyan et al.,1998) for details the method). The ability of peptides to dissociate apreformed hetero-trimer was assessed by comparing the rates of Fα_(i1)dissociation from Fα_(i1)β₁γ₂ with excess unlabeled α_(i1) competitor(intrinsic dissocia-tion rate) and in the presence of a saturatingconcentration of peptide. In FIG. 1C, it can be seen that in thepresence of SIGK the rate of dissociation of Fα_(i1) is faster than theintrinsic rate of dissociation, (i.e. with excess α_(i1)) while withSCAR the rate is the same as the intrinsic rate. Thus two peptides thatbind to the same interface but use unique contacts within the interfacehave two different effects on subunit dissociation.

In summary, these data reinforce the idea that Gβγ functions can beselectively altered by targeting the “hot spot” with peptides. The factthat the selective peptides that were developed bind to the “hot spot”switch II interface, indicates that reagents that bind this surface canbe selective for Gβγ subunit interactions. Thus, for SIGK, theselectivity for effectors results from some Gβγ targets having keyinteractions outside the “hot spot” (Scott et al., 2001). The new datademonstrating that peptides have unique binding determinants within thehot spot that may dictate selectivity for α subunit interactionsindicates that selectivity can be achieved within the “hot spot” thatcan be pharmacologically exploited to develop selective drugs.

(2) Virtual Screening of the NCI Diversity Library.

Given the ability to develop peptides selective for G protein functionswithin a single binding site, when small molecules are found that bindwithin this site, an even higher level of selectivity is achieved. Sincepeptides are relatively large, it can be more difficult to selectivelysterically modify protein-protein interactions within the hot spot. Tofind small molecules that bind to this site the NCI diversity library of1990 compounds was obtained from the Developmental Therapeutics programat the National Cancer Institute that represents a larger library of250,251 compounds. A virtual version of this library was screened forbinding to the Gβγ “hot spot” using Syby1/FlexX virtual dockingsoftware. For the purposes of this screen the “hot spot” was defined asthe surface area of Gβγ within 6.5 Å of the peptide binding site (FIG.3A). FlexX computationally poses the molecule in the “hot spot” and eachof the selected poses is evaluated with five scoring functions (D-score,G-score, F-score, chemscore, and PMF score (Wang et al., 2003) and twoconsensus scores. Eighty five compounds representing the top 1% fromeach scoring function were selected and tested for interaction with Gβγin the competitive phage ELISA described below.

(3) Selected Compounds Inhibit SIGK Peptide Binding.

Here, compounds identified as potential binders computationally weretested to determine whether they would compete for binding of a phagedisplaying SIGK. Since the binding of SIGK to Gβγ has been structurallydefined, compounds that compete for SIGK binding were selected ascompounds that bound to the “hot spot” on Gβγ. The phage ELISA assay wasused to rapidly screen the ability of compounds to bind to the proteininteraction “hot spot” based on the method of Scott et. al (Scott etal., 2001). This assay is performed in the presence of detergent (0.5%Tween) to eliminate non-specific compound aggregation artifacts. Becauseof the assay's simplicity, compounds that influence the binding arelikely to be directly affecting βγ. Compounds that blocked SIGK-phagebinding were then titrated to determine their affinity for Gβγ (FIG.3B). Initial screening identified 9 candidate compounds that inhibitedSIGK binding with IC₅₀'s ranging from 100 nM to 60 μM (Table 18).

TABLE 18 Compounds identified in Elisa screen Compound (NSC#) MW IC₅₀ μMM119 (119910) 370 Da 0.2 M306 (306711) 1001 Da  7 M308 (308820) 319 Da0.1 M117 (117079) 437 Da 56 M121 (12155) 445 Da 13 M231 (23128) 899 Da16 M402 (402959) 779 Da 2 M125 (125910) 566 Da 18 M109 (109268) 679 Da 4

(4) Structure Activity Relationships.

One compound, M119, with high “apparent” affinity for Gβ₁γ₂ (ELISAIC₅₀=200 nM) (FIGS. 3B and C) was selected as a lead to definestructure-activity requirements (SAR) for binding to Gβ₁γ₂. M308, whichalso had a very high “apparent” affinity for Gβγ was found to act via aβγ-dependent oxidation mechanism and was not pursued further in thisanalysis. The NCI library was searched for compounds similar to M119using Syby1/Unity data base searching software. This software creates atwo dimensional chemical fingerprint, taking into account the number ofhydrogen bond donors and acceptors and other physiochemical parameters,and used this fingerprint to search the 250,000 compound NCI library. 21compounds from the NCI library with similar structures to M119 weretested for relative Gβ₁γ₂ binding affinities (See FIG. 18 forrepresentative binders). For example, the apparent affinity of M119B forGβ₁γ₂ is 1/1000 that of M119 with the key chemical difference being theloss of 2 hydroxyl groups (FIG. 3C). Thus, specific chemicalcharacteristics are required for interactions with the Gβγ hotspot.

(5) α-Subunit Interactions.

While the compounds inhibited interactions between Gβ₁γ₂ and the peptideSIGK, it is thought to be relatively difficult for small compounds todisrupt true protein-protein interactions. Whether M119 could disruptprotein interactions was tested with a bona fide Gβγ binding partner,Gα_(i1). The ‘hot spot’ for protein interaction overlaps with the Gαswitch II binding surface on Gβγ. The overall Gα_(i1)-βγ interactionsurface spans 1800 Å² (Lambright et al., 1996; Wall et al., 1995) andthe dissociation constant (K_(d)) for Gα_(i1) binding to Gβγ isapproximately 1 nM (Sarvazyan et al., 1998). M119 competed withFITC-α_(i1) (Fα_(i)) for binding to bGβ₁γ₂ with an IC₅₀ value of 400 nM(FIG. 3D). However, unlike SIGK and related peptides that bind to thissurface (Ghosh et al., 2003), M119 did not promote dissociation ofGα_(i) from Gβγ (Bonacci et al., 2006).

(6) Effect of Compounds on in vitro PI3Kγ, PLCβ2/3 and GRK2 Regulationand Binding by Gβγ.

FlexX docking software predicted that compounds M201 and M119 (FIG. 3C)bound to distinct subsurfaces in the ‘hot spot’, but M201 did notcompete for SIGK binding. Nevertheless, M119 and M201 were tested in invitro reconstitution assays of Gβγ-dependent activation of PLCβ2, PLCβ3and PI3Kγ and binding to GRK2. M119 attenuated Gβ₁γ₂-dependentactivation of PLCβ2 (IC₅₀ value of 3 μM), PLCβ3 and PI3Kγ (FIG. 4A-Cleft panels). M201, on the other hand, did not affect PLCβ2 activationby Gβ₁γ₂ but potentiated Gβ₁γ₂-dependent activation of both PLCβ3 andPI3Kγ (FIGS. 4A-C right panels). M119 also inhibited direct binding ofbGβ₁γ₂ to PLCβ2 and PLCβ3, while M201 did not block binding of PLCβ2 andenhanced binding of PLCβ3 to Gβ₁γ₂ (FIGS. 4E and F). M119 and M201 bothinhibited GRK2 binding to bGβ₁γ₂ with similar IC₅₀ values ofapproximately 5 μM (FIG. 4D). A weakly binding compound M119B (FIG. 3C)did not have effects in these assays (Bonacci et al., 2006). These datashow that small molecules differentially modulate Gβγ interactions witheffectors. These are only two of multiple diverse compounds identified,indicating potential for multiple modes of Gβγ-dependent targetmodulation by these small molecules.

TABLE 19 ELISA PLCb2 PLCb3 PI 3-Kg Compound (μM) (μM) (μM) (μM) M119 0.23 3 8 115360 (M360) 12 12 75 2.5 115372 (M372) 19 14 15 6 402959 (M402)2 NE NE 12 23128 (M231) 27 167 NE 13.5 Summary of effector IC50's. n ≧ 2(each in duplicate). NE = No effect.

Table 19 shows the IC50's of compounds found to be relatively selectivefor inhibition of βγ-dependent regulation of PI3Kγ relative toregulation of PLCβ23 and β3. Compounds were tested for βγ-dependentregulation of these enzymes in in vitro enzyme assays as describedearlier.

(7) Biophysical Analysis of Ligand Binding by SPR.

The competition ELISA is a valuable medium throughput method thatimplies efficacy and localizes the binding site, but the apparentK_(d)'s estimated from IC₅₀ values in the ELISA assay do not give a truemeasure of binding affinity because it relies on competition withmultivalent peptide binding. To understand the protein bindingcharacteristics of compounds a surface plasmon resonance (SPR) methodwas developed for monitoring direct small molecule binding to G βγsubunits using a Reichert SPR instrument. In addition to bindingaffinity, binding kinetics can be obtained from this analysis. Given anIC₅₀ in the range of 1 μM, it was suspected that M119 binds withrelatively rapid on/off kinetics. To obtain the data in herein, 3000refractive index units (RIU) of Gβ₁γ₂ was immobilized to maximizedetection of small molecule binding (Karlsson et al., 2006; Markgren etal., 2001; Rich et al., 2001) and compounds were injected at 75 μl/minand changes in bulk refractive index were accounted for in a referenceflow cell. No non-specific binding of the compound to the sensor surfacein the absence of Gβγ was detected. In the analysis of M119 binding toGβγ SPR, the results indicate that k_(on) and k_(off) are much slowerthan expected (global fitting analysis gave estimated k_(on) 2×10³M⁻¹s⁻¹, k_(off) 2×10⁻⁴ s⁻¹, K_(d) 100 nM). This data demonstrates thatsmall molecule binding to Gβγ by SPR can be detected and analyzed. Thismethod is used to analyze compound binding kinetics and calculateequilibrium binding constants to test the mechanisms of action andselectivity of various “hot spot” binding compounds.

(8) Analysis of Compound Efficacy and Selectivity in Intact Cells.

To test the effects of differentially targeting Gβγ on GPCR signaling inintact cells, M119 (and a similar compound M158C) (See FIG. 3C) and M201were tested for their ability to modulate fMLP receptor-dependentsignaling in differentiated HL60 leukocytes. The fMLP receptor couplesto Gi in these cells and activates PLCβ2 (PLCβ3 is a minor isoform inthese cells), PI3Kγ, and ERK through Gβγ signaling (Li et al., 2000;Neptune and Bourne, 1997). Pre-treatment of differentiated HL60 cellswith M119 and M158C (FIGS. 5A), but not M201 (FIGS. 5C and D),attenuated fMLP-induced Ca²⁺ increases. M119 had no effect oncarbachol-dependent increases in Ca²⁺ in HEK293 cells stably expressingthe Gq-linked M3-muscarinic receptor, confirming a specific effect ofM119 on Gβγ-dependent Ca²⁺ mobilization (FIG. 5B). fMLP-dependent GRK2translocation to the membrane fraction of HL60 cells on the other handwas substantially inhibited by incubation with either M119 or M201 (FIG.5E). Thus M119 and M201 differentially modulate PLCβ2 regulation by Gβγ,yet both inhibit GRK2 binding in intact cells.

To assess the ability of M119 and M158C to inhibit fMLPreceptor-dependent regulation of PI3Kγ activation, HL60 cells werestimulated stably over-expressing a GFP tagged PH domain from Akt(Servant et al., 2000) with fMLP, and assessed translocation of theGFP-PHAkt to the membrane by subcellular fractionation and Westernblotting. The PH domain from Akt binds to PIP₃ produced by PI3K activityat the membrane. Pre-treatment of the cells with 10 μM of M119 or M158Cinhibited fMLP-dependent translocation of GFP-PHAkt to the membrane(FIG. 5F), consistent with the ability of these compounds to inhibitactivation of PI3Kγ by Gβγ.

Stimulation of differentiated HL60 cells with fMLP also results inpertussis toxin sensitive activation of various MAP kinases includingERK1 and ERK2, p38 and JNK (Rane et al., 1997). However, pretreatment ofHL60 cells with M119, M158C or M201 did not block fMLP-inducedactivation of ERK1 and ERK2 (FIG. 5G).

(9) Characterization of Novel Compound Binding to Gβγ

Previous studies identified multiple compounds that blocked effectorbinding to Gβγ. A lead compound, M119, inhibited Gβγ-dependentPI3-kinase γ and PLCβ activation in vitro and blockedchemoattractant/PI3-kinase-dependent GFP-PH-Akt translocation tomembranes as well as Ca2+ release (Bonacci et al., 2006). A relatedcompound, galleon (DL382), was identified that differs from M119 by thesubstitution of a benzene carboxylic acid for cyclohexane carboxylicacid at the 9 position of the core xanthene (FIG. 8A). Gallein iscommercially available at high purity as a single isomer and inquantities necessary for in vivo analysis, so the Gβγ binding propertiesof gallein were compared with M119. Gallein effectively competed forbinding of SIGK peptide to Gβγ in a phage ELISA assay with an IC50comparable with that of M119 (FIG. 8B). Herein is shown that compoundsthat block peptide binding in this assay are effective competitors ofmany Gβγ protein-protein interactions.

To determine direct binding equilibrium and kinetic constants forgallein binding to Gβγ, surface plasmon resonance (SPR) measurementswere performed with streptavidin-immobilized biotinylated Gβ1γ2. Bindingand dissociation of gallein was monitored as a function of concentrationand time. Data were fit to one site association and dissociation modelsto calculate association and dissociation rate constants from whichaffinity constants were derived (FIG. 8C, Table 21). Based on the SPRanalysis, gallein bound to Gβ1γ2 (FIG. 8C) with a Kd value ofapproximately 400 nM (Table 21), in relatively close agreement with theIC50 of 200 nM observed in the competition ELISA assay. The controlcompound, fluorescein, which did compete for SIGK binding in thecompetition ELISA, did not have detectable binding by SPR (Table 21). Itis noteworthy that binding and dissociation rates for gallein wererelatively slow (FIG. 8C). These data confirm that gallein bindsdirectly to Gβγ, and resulting effects on competition for effectorbinding are likely to result from direct binding to Gβγ with highaffinity. Based on these data, gallein has similar effects to M119 whentested in in vitro and in vivo assays.

(10) Inhibition of G Protein-Dependent Chemotactic Peptide Signalingwith Small Molecules

As discussed in background and significance Gβγ-dependent activation ofPI3kγ in neutrophils is important in directing neutrophil migration inresponse to chemoattractants. Activation of this receptor system leadsto a gradient of PIP₃ production with enhanced accumulation at theleading edge of the cell that is important for polarizing the cells inthe direction of the chemo-attractant (Servant et al., 2000; Wang etal., 2002). In animal models of neutrophil chemotaxis, deletion of PI3kγresults in defects in neutrophil accumulation and reduced inflammation(Hirsch et al., 2000; Li et al., 2000). To determine if blockingGβγ-depend-ent activation of PI3Kγ in neutro-phils would affectchemotaxis, the Boyden chamber assay was used to quantitate chemotaxis.fMLP enhanced migration and inclusion of 10 μM M119 with fMLPsignificantly blunted migration (FIG. 10E), supporting the idea thatblocking Gβγ dependent signaling (likely PI3K activation) in HL60 cellsinhibits migration. The same assay with M119 without fMLP did not inducemotility and the inactive control M119B had no effect on migration. Todetermine whether galleon (DL382) (see FIG. 8 for structure), like M119,modulates the receptor-dependent activation of PI3-kinase, HL60 cellsexpressing GFP-PH-Akt were pretreated with compound and challenged withfMLP. Gallein inhibited fMLP-dependent GFP-PH-Akt translocation indifferentiated HL60 cells with an efficacy comparable with that of M119(FIG. 9A).

Activation of Rac in neutrophils is critical for fMLP-dependentactivation of NADPH-oxidase and subsequent superoxide production and isdependent on Gβγ- and PIP₃-dependent activation of the Rac-specificexchange factor P-Rex. Herein is shown that M119 inhibited P-Rex1activation by fMLP suggesting the compounds could inhibit Rac activation(Zhao et al., 2007). It was determined whether Gβγ-binding compoundswould inhibit receptor-dependent activation of Rac1. Rac1 GTP levelswere measured in cytosolic extracts of HL60 cells that had beenpretreated with compounds before stimulation with fMLP. M119 and galleininhibited fMLP-induced Rac1 activation in differentiated HL60 cells,whereas the negative control compound M119B had little effect (FIG. 9B).

(11) Gβγ Inhibitors Blocked fMLP-Dependent Superoxide Production andNeutrophil Chemotaxis.

The ability of these small molecules to block fMLP-induced chemotaxisand superoxide production was assessed to determine whether thecompounds can block relevant cellular functions downstream of PI3-kinaseγ and Rac. Both of these functions are critical to the inflammatoryprocess and inhibition of both processes can contribute toanti-inflammatory effects of Gβγ inhibitors.

M119 and gallein both significantly inhibited fMLP-dependent superoxideproduction. Control compounds M119B and fluorescein did notsignificantly affect this response (FIG. 9C). Wortmannin also blockedfMLP-dependent superoxide production, indicating that the pathway was aPI3-kinase-dependent pathway. PMA-dependent superoxide production, aprocess not dependent on Gβγ was also examined. PMA-dependent superoxideproduction was not inhibited by any of the Gβγ binding compounds orcontrols (FIG. 9D), indicating that the compounds specifically inhibitedthe Gβγ-dependent pathway to superoxide production.

fMLP-dependent chemotaxis was assessed in a Boyden Chamber. M119 andgallein inhibited fMLP-induced chemotaxis in differentiated HL60 cellswith similar efficacy (FIG. 10A). Neither M119 nor gallein had anyeffects on chemokinesis measured in the transwell assay with fMLP inboth the upper and lower chambers (data not shown). To support the ideathat the mode of action of these compounds is dependent on Gβγheterodimers liberated from Gα_(i)-coupled chemokine receptors, cellswere challenged with GM-CSF. Chemotaxis induced by GM-CSF is partlydependent on PI3-kinase activity in human neutrophils but is independentof Gβγ-stimulation of PI3-kinase (Gomez-Cambronero et al., 2003). If thesmall molecules were acting directly on PI3-kinase or by a nonspecificmechanism, they would be expected to block GM-CSF-induced chemotaxis.Neither M119 nor gallein blocked GM-CSF-induced chemotaxis indifferentiated HL60 cells (FIG. 10B). These data demonstrate two keypoints: 1) the general chemotaxis machinery in HL60 cells is notaffected by these compounds and 2) the compounds selectively inhibitedGPCR-dependent chemotaxis, consistent with inhibition of Gβγ signaling.

The inhibitory properties of these compounds in potentially moreclinically relevant isolated primary human neutrophils were alsoevaluated. Again, both M119 and gallein significantly inhibitedfMLP-induced chemotaxis (FIG. 10C). Wortmannin, a general PI3-kinaseinhibitor (Okada et al., 1994), also blocked chemotaxis (FIG. 10C).Conversely, M119B binds only weakly to the Gβγ “hot spot” (Table 21 inBonacci et al., 2006) and had no affect on cell motility (FIG. 10C),suggesting that the observed effects on chemotaxis are dependent onsmall-molecule-binding to the Gβγ “hot spot.” IL-8 regulated chemotaxiswas also blocked by M119 and gallein (FIG. 10C) extending the findingsto other G_(i) coupled chemoattractants and supporting the idea that themechanism of action of these compounds is to inhibit Gβγ subunitsignaling down-stream of chemoattractant/chemokine receptors.

To assess the potency of gallein for inhibition of chemotaxis, primaryhuman neutrophils were treated with a range of concentrations andassayed for fMLP-dependent chemotaxis. Gallein blocked fMLP-dependentchemotaxis with an IC₅₀ of approximately 5 μM (FIG. 10D). Thus, smallmolecules that bind to Gβγ and block Gβγ-dependent PI3-kinaseγregulation in vitro and in HL60 cells potently inhibit GPCR-dependentneutrophil chemotaxis with an IC₅₀ comparable with what has beenpublished for direct PI3-kinase catalytic inhibitors onchemokine-dependent monocyte chemotaxis (Camps et al., 2005).

(12) Gallein Attenuates Inflammation and Neutrophil Recruitment in Vivo.

Inhibition of chemoattractant-dependent chemotaxis of neutrophils wouldbe predicted to inhibit neutrophil-dependent inflammation based on datafrom PI3-kinase γ knock-out mice (Hirsch et al., 2000; Li et al., 2000).To assess the in vivo efficacy of Gβγ-binding small molecules inblocking neutrophil chemotaxis gallein was tested in the carrageenan pawedema model. Carrageenan, when injected into the glabrous tissue of thehind paw, leads to rapid acute inflammation characterized byinfiltration of neutrophils (Siqueira-Junior et al., 2003). Gallein orvehicle control was delivered by intraperitoneal injection 1 h beforeinjection of carrageenan into the paw. Peak paw edema was observed 2 hafter carrageenan injection (FIG. 11A). Indomethacin, a nonselectivenonsteroidal anti-inflammatory drug that inhibits cyclooxygenases 1 and2 (Siqueira-Junior et al., 2003), is the drug of choice for comparativein vivo efficacy studies. Pretreatment with indomethacin sharply reducedpaw edema. It is noteworthy that prophylactic administration of 100mg/kg gallein reduced paw edema to levels comparable with that ofindomethacin. Injection with gallein in the absence of carrageenan hadno effect on paw thickness (data not shown). Additional experimentationdetermined that the ED₅₀ value of gallein for inhibiting paw edema to beapproximately 20 mg/kg (FIG. 11B). Acute phase inflammation, as seen inthe carrageenan-induced paw edema model, is characterized by neutrophilinfiltration (Siqueira-Junior et al., 2003; Posadas et al., 2004). Toconfirm that neutrophil infiltration was inhibited, the number ofneutrophils in paw exudates was quantified. Pretreatment with 100 mg/kggallein reduced the number of neutrophils in the edematous fluid byapproximately half to levels comparable with that seen with indomethacin(FIG. 11C). These data correlated well with the actual volume of exudatefluid. Pretreatment with indomethacin and gallein also reduced exudatevolume by approximately 75% (FIG. 11D).

Oral administration of gallein (30 mg/kg) in mice 1 h before carrageenanchallenge also significantly reduced paw swelling by 40% comparable withthat of indomethacin (FIG. 12). These data demonstrate that gallein isabsorbed into systemic circulation, is bioavailable, and systemicallyimpairs neutrophil recruitment. To support the conclusion that theobserved reduction in paw swelling was Gβγ-dependent, the M119B-likecompound fluorescein was tested under identical experimental conditions.Fluorescein differs from M119B only by the substitution of an aromaticbenzene ring for a cyclohexane at the 9 position of the core xanthene(FIG. 8A) and binds Gβγ very weakly if at all (Table 21). Fluoresceindid not reduce paw swelling under identical experimental conditions(FIG. 12), which indicates that the observed reduction in paw swellingand neutrophil infiltration was correlated with the ability of the smallmolecules to bind to Gβγ. Based on this data and other publishedstructure activity data one key structural requirements for activityresides in the core xanthene scaffold with requirements forhydroxylation at the 3,4,5 and 6 positions of the xanthene ring system.

g) Discussion

Herein a novel strategy for treatment of inflammation that targetsinteractions between G protein βγ subunits and effectors that arecritical for neutrophil migration in response to activation ofchemoattractant receptors was presented. Gβγ-responsive PI3-kinaseγproduction of PIP₃ is critical to neutrophil functions, includingsuperoxide production, chemotaxis, and cellular polarization (Hirsch etal., 2000; Li et al., 2000). It was shown that Gβγ-binding smallmolecules inhibit interactions between Gβγ and PI3-kinase γ. These samemolecules block PI3-kinase and Rac1 activation in HL60 cells,chemoattractant-dependent superoxide production and chemotaxis indifferentiated HL60 cells. These findings were extended to inhibition ofchemoattractant-dependent chemotaxis in primary human neutrophils andultimately neutrophil-dependent inflammation in vivo.

PI3-kinases play diverse roles in normal cellular physiology, includingcell motility and survival (Cantley, 2002). Camps and colleaguesidentified and characterized small-molecule inhibitors of PI3-kinase γthat are competitive with ATP (Camps et al., 2005). These smallmolecules were efficacious in mouse models of rheumatoid arthritis andsystemic lupus, providing further rationale for pharmacologicaltargeting of the pathway as therapeutic strategy (Barber et al., 2005;Camps et al., 2005). However, a concern with inhibitors of this class isthat because many kinases of the same family have significant homology,there may be difficulty in developing inhibitors that are selective forPI3-kinase γ, although some progress has been made in this area (Rückleet al., 2006). The strategy presented here selects for a single isoformof the PI3-kinase family because PI3-kinase γ is the only PI3-kinaseisoform that uses G_(i)βγ-dependent activation as a major mechanism forregulation (Stephens et al., 1994, 1997; Hirsch et al., 2000).

Compounds such as M119 and gallein also inhibit interactions between Gβγand other targets that are critical for chemoattractant-dependentdirected migration or reactive oxygen species production by neutrophilsor other monocytes. For example, M119 blocks membrane translocation ofP-Rex, a PIP₃- and Gβγ-regulated Rac2 exchange factor, in humanneutrophils, which is due in part to directly blocking P-Rex binding toGβγ in addition to blocking PIP₃ production by PI3-kinase (Zhao et al.,2007). It has recently been shown that Ras is required for fullPI3-kinase γ activation in neutrophils (Suire et al., 2006). It ispossible that the inhibitors block Gβγ-dependent activation of Ras andin part act to block PI3-kinase γ activation in cells indirectly throughinhibition of Ras activation. In Dictyostelium discoideum, bothPI3-kinase and phospholipase A₂ activities downstream of G proteinactivation are important for chemotaxis (Chen et al., 2007). Otherstudies have shown chemoattractant-dependent chemotaxis becomesPI3-kinase-independent under certain conditions (Ferguson et al., 2007).Thus, a broad-spectrum Gβγ inhibitor is more effective, in some cases,than selective PI3-kinase γinhibitors because they can block other Gβγinteractions besides PI3-kinase-γ, a property that contributes totherapeutic efficacy. Nevertheless, because of the wide roles of Gβγ inregulation of cell physiology, development of Gβγ binding smallmolecules that are more selective for PI3-kinase γ inhibition relativeto other Gβγ-dependent pathways is an important direction.

Inflammation is a central mediator of many human conditions, includingatherosclerosis, allergic reactions, psoriasis, virus-inducedmyocarditis, ischemia-reperfusion injury, and rheumatoid arthritis. Theinflammatory process involves complex signaling cascades partlycoordinated by chemokines, which recruit leukocytes, including largenumbers of neutrophils, to sites of inflammation (Wu et al., 2000).Targeting chemokines with antibodies or binding proteins as well astargeting chemokine receptors has been attempted as a therapeuticstrategy (Gong et al., 1997; Ogata et al., 1997; Plater-Zyberk et al.,1997; Barnes et al., 1998; Halloran et al., 1999; Matthys et al., 2001;Podolin et al., 2002; Yang et al., 2002) However, the overwhelmingcomplexity of these signaling molecules (multiple chemokines, chemokinereceptors, and redundancy) is a significant hurdle. Polychemokine(Carter, 2002) or combinations of different chemokine (al Mughales etal., 1996) antagonists have been suggested, but there are chemokinesthat act as an agonist at one receptor and an antagonist at another(Xanthou et al., 2003). Despite this complexity, these chemokinereceptors and ligands represent a tantalizing therapeutic target becauseof their integral role in the inflammatory process. Targeting PI3-kinaseγ has been suggested as strategy for blocking a common signal downstreamof chemokine receptors (Camps et al., 2005; Rückle et al., 2006). Thedata presented herein indicate that targeting Gβγ also circumvents theredundancy of the chemokine system and can offer some advantages todirect targeting of PI3-kinase γ.

h) Additional Methods

(1) Analysis of Selectivity of Gβγ-Binding Compounds.

Herein, the selectivity of G protein βγ-binding compounds is examined at3 levels of G protein signaling: 1) effector selectivity, 2) effects onGPCR coupling to G protein-dependent nucleotide cycling, and 3)selectivity for G protein βγ subtypes. All of these factors contributeto the potential utility of this novel class of compounds as therapeuticagents. The data demonstrate the therapeutic efficacy of this approachwith a relatively general inhibitor of Gβγ-effector function (M119), butthe more selective a particular compound for a particular function, thegreater the potential for selective efficacy in vivo. These studiesprovide the critical basis for evaluating the significance of thebiophysical measurements. Understanding the nature of the selectivity isimportant for evaluating structurally how selectivity is generated at amolecular level.

A second series of experiments involves development of a new assay thatprovides a rapid method for analysis of selectivity without testing eachcompound in individual reconstitution assays. After fully characterizingthe selectivity and efficacy of these compounds on various G proteineffector systems, the results are correlated with selectivity defined intwo rapid screening assays. These assays provide a tool for rapidprediction of effector selectivity. Since inhibition of specificeffector systems may correlate with therapeutic utility (for example,inhibition of Gβγ-dependent PI3Kγ regulation might be useful in treatinginflammatory diseases), this analysis provides a faster approach foridentification of compounds with specific therapeutic predictabletherapeutic applications.

(2) Effector Specificity of Gβγ Binding Compounds.

Disclosed herein, a focused comparison of M119 and M201 was presented inGβγ-effector selectivity to demonstrate the concept that effectorselectivity can be achieved with this small molecule approach. Here,this analysis is extended to other effector systems and to othercompounds that have not been tested for specificity.

(3) New Compounds.

Several other lead compounds (besides M119 and M201) were identifiedthat block SIGK binding to Gβγ in the low to mid μM range (Table 18).The effector selectivity of these molecules is characterized as was donewith M119 and M201, and further as described below for new effectorsystems. It is understood and herein contemplated that uniquespecificity profiles for different compounds are identified. Since manyof these compounds appear to bind in the low μM range, it is desirableto identify related compounds that bind with higher affinity. Asdiscussed for M119, similarity searches were performed for all of thelead compounds in Table 18 to identify compounds structurally related tothe lead compounds in the NCI library. These compounds were obtainedfrom the NCI and tested in the phage ELISA and by SPR binding analysisas described herein. To control for nonspecific effects compoundscontaining metal ions are avoided, and activities are measured in thepresence reducing agents such as DTT and metal chelators such as EDTA.For example, it was found that the activity one of the compounds inTable 18, M308 is blocked by DTT and is thus is likely acting via aredox mechanism, and it is no longer be considered in this analysis.Thus, a family of compounds with unique specificity profiles that bindwith relatively high affinity is collected.

(4) New Effectors.

Experiments presented herein focused on compound selectivity forGβγ-dependent PLCβ2, PLCβ3, PI3Kγ and GRK2 as proof of principle. Theeffector specificity analysis can be expanded to other majorGβγ-dependent effector systems including adenylyl cyclase type I andtype II, N-type Ca²⁺ channels, inwardly rectifying potassium (GIRK)channels and ERK activation.

Compound effects on Gβγ-dependent regulation of ACI and ACII are testedusing Sf9 cell membranes expressing either of these two isoforms aspreviously described (Scott et al., 2001; Taussig et al., 1994). ACIexpressing membranes (10 μg) in the presence of forskolin, and ACIIexpressing membranes in the presence of added 50 nM GsαGTPγS, with orwithout 100 nM Gβ₁γ₂ subunits, are assayed for cAMP production.Compounds are titrated around their apparent K_(d) estimated from theirELISA IC₅₀'s. When the compound affects forskolin (ACI) or Gsα (ACII)stimulated activity in the absence of Gβγ the effects are considerednon-specific and are be pursued further.

Effects on GIRK channels and N-type Ca²⁺ channels are assessed inprimary rat superior cervical ganglion (SCG) neurons (Kammermeier etal., 2000; Ruiz-Velasco and Ikeda, 1998). Norepinephrine inhibits N-typeCa²⁺ channels through a Gβγ dependent mechanism in SCG neurons andactivates GIRK channels when they are heterologously expressed. Neuronsare pretreated with varying concentrations of compounds (either in thebath or included in the patch pipette), and NE dependent inhibition ofN-type Ca²⁺ channels are measured in the whole cell patch clampconfiguration. Initial analysis indicates that this pathway isunaffected by M119. When compounds are found that block inhibition ofCa²⁺ channels, it is confirmed that the effects are reproduced in aprepulse facilitation protocol in the presence of heterologouslyexpressed Gβγ to confirm that the compounds are acting at the level ofGβγ. N-type Calcium currents (CaV 2.1) directly inhibited by Gβγ exhibitslowed activation kinetics and voltage dependence such that strongdepolarizing steps can partially reverse the inhibition. This voltagedependence is diagnostic of Gβγ-mediated modulation. The “facilitation”ratio of the postpulse current to the prepulse current (during whichinhibition is strong) provides a quantitative measure of the degree ofGβγ modulation. Uninhibited currents generally have a facilitation ratioaround 1 so cells pretreated with compounds that inhibit Gβγinteractions with the channel causes the facilitation ratio toapproach 1. The effects on responses to other receptors such astransfected μ-opioid receptors or endogenous M4 muscarinic receptors arealso examined. GIRK channel regulation is assessed by microinjectingGIRK1/2 subunits in SCG neurons and measuring K⁺ channel regulation byNE at varying concentrations of compound. Basal K⁺ channel activity andCa²⁺ channel activity is assessed to look for non-specific effects ofGβγ blockers. Outcomes from these experiments are that the compoundsblock both of these currents or compounds are identified thatselectively modulate one or the other of the currents. When compoundshave no effect on either Ca²⁺ channel or K⁺ channel regulation, this isinteresting and indicates that the compound inhibits some effectors butnot these channels.

Gβγ-dependent ERK activation is measured in two cellular systems thatwere examined previously using both a physiological agonist and a directactivator of Gβγ signaling. In primary rat arterial smooth muscle cells(RASM) and in Chinese hamster ovary (CHO) cells, lysophosphatidic acid(LPA) activates ERK in a PTX-sensitive manner that involves Gβγ. In RASMcells LPA promotes cell division in culture, and vascular restenosis invivo in a Gβγ- and ERK-dependent manner (Iaccarino et al., 1999). A cellpermeable Gβγ-binding peptide (mSIRK) that activates ERK signaling inboth cell types through a nucleotide exchange independent G proteinsubunit dissociation mechanism (Goubaeva et al., 2003; Malik et al.,2005) are also tested. This peptide allows assessment of Gβγ-dependentERK activation more directly than with LPA. Cells are treated withcompounds at their apparent IC₅₀ or above for 10 min followed byaddition of 5 nM LPA or 10 μM mSIRK. Control, inactive compounds such asM119B are used where appropriate and cells treated with compound alone(no LPA or mSIRK) are also analyzed. ERK activation is measured withp-42/p44 phospho-ERK immunoblotting. Compounds found to affect ERKactivation are further characterized for dose dependence andspecificity.

(5) Effects of Compounds on Receptor-G Protein Coupling.

Receptor coupling to G proteins requires interaction between G protein αand βγ subunits. M119 blocks interaction between Gα and Gβγ subunits inan equilibrium binding assay (See FIG. 3D), but does not promote subunitdissociation. Compounds that bind at the Gα/βγ interface also interferewith receptor-dependent G protein activation, yet this does not appearto be the case in vivo or in cells since some aspects ofreceptor-effector coupling appears to be intact in systems where M119 isotherwise blocking Gβγ functions (FIGS. 5G and 14A). A receptor-Gprotein reconstitution system is used to determine if the differentcompounds alter the ability of receptors to stimulate GTPγS binding to Gprotein α subunits or GTP hydrolysis by α subunits. The receptorreconstitution systems developed by John Northup (Hellmich et al., 1997)and Peter Chidiac (Cladman and Chidiac, 2002) are used to accomplishthis. The system developed by Northup measures [³⁵S]-GTPγS binding topurified G protein subunits added back to urea stripped Sf9 cellmembranes expressing specific GPCRs, while the system developed byChidiac examines GTP hydrolysis by G proteins coexpressed with receptorsin Sf9 membranes. Both GTPγS binding and GTP hydrolysis assays areimportant for this analysis. GTPγS binding is dependent on a singleround of G protein activation while GTP hydrolysis can involve severalrounds of GTP binding and hydrolysis. Compounds like M119 do notinterfere with receptor-dependent GTPγS binding because the compounddoes not have access to the binding site in the heterotrimer and becauseno reassociation of Gα and Gβγ is required in the single round ofnucleotide exchange. With receptor stimulated GTP hydrolysis, there aremultiple rounds of dissociation and reassociation. Under theseconditions M119 or other compounds gain access to the Gβγ/α interfaceand inhibit receptor stimulated GTP hydrolysis. This is dependent uponthe kinetics of G protein dissociation and reassociation as well as theon and off rates for compound binding to Gβγ.

Sf9 insect cells are infected with viruses expressing M2 muscarinicreceptors (or other receptors as below), membranes are prepared fromthese cells and the endogenous G proteins are inactivated by treatmentwith 7M urea. Purified Gα_(i1) and β₁γ₂ proteins are reconstituted withthe membranes, and carbachol and Gβγ-dependent binding of [³⁵S]-GTPγS toGα_(i1) are measured in the presence of multiple concentrations ofcompound. It has been previously demonstrated that in this assay, Gβγsubunits are required for agonist stimulated activities (Goubaeva etal., 2003; Hellmich et al., 1997).

Rather than the compounds simply interfering with Gα/βγ interactions,they can also interfere with direct interactions between Gβγ subunitsand receptors. There is evidence for direct interactions betweenreceptors and Gβγ that can influence G protein activation and signaling(Mahon et al., 2006; Taylor et al., 1996; Wu et al., 2000). Coupling ofa variety of receptors to G proteins is assessed in the Sf9 membranereconstitution assay. When compounds interfere with determinants on Gprotein Gβγ subunits that interact directly with receptors, the compoundinhibits receptor dependent-activation but can be selective for certainreceptors. When the compounds act by inhibiting Gα/βγ interactions theyblock activation of all receptors tested. A variety of Gi coupledreceptors are tested including α₂-adrenergic receptors and μ-opioidreceptors and look for selectivity for different receptors. When thereis a selective effect on different GPCRs, it means that the compoundsare interfering with specific interactions between the receptor and theG protein.

(6) G Protein βγ Subtype Selectivity.

A major question in the field of G protein βγ subunit signaling is thesignificance of Gβγ subunit diversity. Multiple G protein βγ subunitcombinations are assembled in vivo although the functional significanceof this diversity is unclear. Many of the amino acids in the “hot spot”are 100% conserved between different G protein β subtypes. Since the“hot spot” was targeted with the screen, no subtype selectivity ispredicted. When there was selectivity for subunit subtypes, in vivoselectivity is much greater. The simplest approach is to use SPR tomeasure interactions of specific compounds with purified G protein βγsubtypes. Specific Gβγ subtypes are purified from Hi5 insect cells.Purified Gβ₁γ₂, Gβ₂γ₂, Gβ₁γ₅ and Gβ₁γ₇ have been acquired. Other Gβγcombinations containing Gβ₃ and Gβ₄ are also purified. These areimmobilized on a biosensor chip via primary amine coupling as describedherein, and individual compounds are titrated, on and off rate constantsdetermined and K_(d)'s calculated from SPR data as described in specificaim 2 and compared between the different Gβγ subtypes.

(7) Global Prediction of Gβγ-Dependent Pathway Specificity.

The goal of this subaim is to develop methods to determine Gβγ-dependentpathway selectivity of individual compounds in a single assay ratherthan testing each compound in individual effector assays. Disclosedherein are two alternative approaches to this.

(a) Signatures in the Peptide Competition Assay.

Here the phage ELISA assay is used to explore compound inhibitionsignatures which are correlated with known effector specificity profilesestablished in in vitro biochemical assays. This approach depends on theobservation that each individual phage displayed peptide has uniquerequirements for specific amino acids in the G protein βγ “hot spot” asdiscussed herein (Davis et al., 2005). When individual compounds eachhave their own unique binding requirements, they differentially affectthe binding of different peptides in a way that is correlated withselectivity for effector binding. Each compound is screened against 9different phage displaying peptides each with unique bindingrequirements as described in (Davis et al., 2005) and obtain aninhibition profile. Compounds are tested at a concentrationcorresponding to their IC₅₀ for inhibition of SIGK phage binding andtheir IC₅₀'s for each peptide are determined. This profile is comparedbetween compounds and the specific inhibition patterns correlated withknown effector profile specificity. For example, groups of compoundsthat are relatively selective inhibitors of Gβγ-dependent PI3Kγregulation have a similar peptide inhibition profile distinct from moreglobal inhibitors of Gβγ function or inhibitors with different effectorselectivities.

(b) Evaluation of Compound Efficacy in a Focused GPCR Array.

Alternatively, gene expression downstream of GPCRs is examined in awhole-cell system to evaluate the effects of selective compounds on GPCRdependent signal transduction. For these experiments a specific GPCRfocused pathway array developed by GE Super Array Bioscience that hasbeen utilized to analyze GPCR signaling pathways is used (Gesty-Palmeret al., 2005; Lee et al., 2005). In this array, 60 mer oligonucleotidesrepresenting 96 human genes known to be regulated downstream of specificGPCR activated pathways have been filter arrayed. The genes have beenselected to represent genes downstream of pathways including Ca²⁺, cAMP,PKC and PI3K. RNA is isolated from stimulated cells and labeled cRNAprobes are created that are hybridized with the array and detected bychemiluminescence imaging. First, this system is used to analyzeselective effects of compounds on signaling downstream of thechemotactic peptide receptor (fMLP) in HL60 human neutrophil like cells.Gene expression profiles in differentiated HL60 cells are analyzed withdifferent doses of fMLP for 5 min, 30 min, 1 h and 4 h to establishconditions with significant alterations in gene expression relevant tokey signaling pathways. Responses of specific pathways are validatedwith specific pathway inhibitors. Wortmannin is used to block PI3Kpathways, U73122 is used to validate PLC responsive genes and H-89 orRp-cAMPs are used to validate cAMP responsive gene expression. With thisassay optimized, the specificity and efficacy of individual Gβγinhibitors is evaluated by adding saturating concentrations of compoundprior to and during fMLP stimulation. Effects of compounds in theabsence of fMLP stimulation are also determined. Specific effects ongene expression are predictable based on their established effectorspecificity profiles.

(8) Determination of the Molecular Basis for Binding and Selectivity ofGβγ Binding Compounds.

(a) Effector Selectivity of M119 Derivatives.

This series of experiments is designed to test the “chemical hypothesis”by examining compounds that bind to the same subsurface of Gβγ but havedifferent chemical characteristics. The premise of this example is thatwhen selectivity is observed for compounds that bind to the samesubsurface of Gβγ it is the chemistry of the compound that isresponsible for the selectivity. Structure activity relationships forcompounds related to M119 were examined in the phage ELISA and it wasfound a series of binding compounds as was discussed herein and shown inFIG. 8. All the active compounds contain a rigid xanthene moiety as acore binding structure and the majority contains hydroxyl groups at the4 and 5 positions that appear to be required for binding. Associatedwith this core structure are a variety of chemical structuressubstituted at the 9 position of the xanthene ring. Since all thesecompounds contain the core xanthene ring and the required 4 and 5hydroxyl groups, they bind to a similar location within the Gβγ “hotspot”. Computational modeling indicates that 5 of these compounds withdiverse substitutions at the 9 position bind to Gβγ at the same site.Thus far, this series of compounds has only been evaluated forcompetition with SIGK binding in the phage ELISA.

Here, the specificity of these compounds predicted to bind to the samesite are examined in the “hot spot” for different effectors as describedherein. Specifically the concentration dependence for each compound isexamined for Gβγ-dependent PLCβ2, PLCβ3, PI3Kγ and GRK2 regulation in invitro reconstitution assays with purified components.

When the compounds all have similar selectivity profiles, it indicatesthat the chemical structure does not strongly influence the selectivityand that steric occupation of specific binding surfaces within the “hotspot” is what drives specificity. On the other hand, when there is adifference in the selectivity profiles for these compounds it indicatesthat the chemical nature of the binding compound presented at theGβγ/effector interface contributes to effector selectivity.

Further evaluation and definition of this concept depends on resultsfrom other assays described herein that more directly evaluate thenature of binding of the compounds to Gβγ. For example, when selectivityfor different effectors is shown by this series of compounds,confirmation that they bind to the same site either by mutagenesis orX-ray crystallography can be performed as described below. Additionally,binding kinetics are evaluated by SPR to determine if differences ink_(on) and k_(off) contributes to the observed specificity.

(9) Evaluation of Ligand-Gβγ Binding Kinetics and Affinities by SPR andITC.

1) The assays used thus far to evaluate the binding of small moleculesto Gβγ have relied on competition analysis to evaluate apparentaffinities. The primary assay has been a phage ELISA competition assaywhere compounds are tested for competition with binding of SIGK peptidebinding to Gβγ. This assay has the advantage that it determines if acompound binds to Gβγ, most likely at the “hot spot” and gives anestimate of apparent affinity from the IC₅₀. In some cases it was foundthat the IC₅₀ in the ELISA correlates well with the IC₅₀ for competitionwith effectors and in other cases it does not.

2) Evaluation of k_(on) and k_(off) allows verification that thekinetics of compound binding contributes to selectivity for Gβγfunction. As discussed herein, the on and off rate constants for M119binding to Gβγ were very slow. This has implications for the mechanismof action of M119 and might form the basis for some level ofselectivity. For example, despite binding at the Gα/βγ interface M119does not disrupt GPCR dependent G protein activation which is thought torequire Gα/βγ interactions. One possible basis for this selectivityrelies on the rapid cycle of subunit dissociation that may occur uponactivation of many G protein systems. M119 may not interfere with thiscycle because the slow k_(on) limits access to the Gα/βγ heterotrimer(Ross and Wilkie, 2000). In cases where free βγ accumulates to asignificant level, this free Gβγ may no longer be participating in Gprotein cycling and is accessible to inhibition by slowly bindingcompounds.

SPR is used to measure binding kinetics and equilibrium bindingconstants for all of the binding molecules identified in the screens andsimilarity searches. This method has the advantage that direct bindingcan be measured to give both the K_(d) and kinetics of the interaction.A second advantage of the method is that relatively low amounts ofprotein are required (relative to ITC for example) which is importantfor analysis of binding to Gβγ mutants as described below. Somedisadvantages include the necessity for immobilization of the targetGβγ, and that binding of low molecular weight molecules is difficult todetect by SPR due to the nature of the method. SPR monitors thealterations in refractive index as a function of mass binding at asurface with immobilized target molecule. As the ligand binds to thetarget the mass bound to the surface increases and changes therefractive index. This method is ideally suited to monitoringprotein-protein interactions because the binding of relatively highmolecular weight materials at the surface causes significant alterationsin mass and thereby refractive index. Lower molecular weight moleculessuch as peptides and low molecular weight organic molecules are moreproblematic because the binding causes only a small change in refractiveindex. Nevertheless, SPR has been used to monitor small moleculeinteractions with immobilized protein and it has been successfullyapplied here. The key to detecting small molecule binding is toimmobilize the target at higher densities than is required formonitoring protein-protein interactions so that significant accumulationof the ligand occurs at the sensor surface.

As discussed herein, an SPR method for binding of small molecule ligandsto Gβγ has ben established. This method was used to measure binding ofthe “hot spot” peptide SIGK. Gβγ was immobilized directly via primaryamine coupling to a hydroxylated surface for this analysis. It hadpreviously been indicated that primary amine coupling of Gβγ to dextrancoated SPR chips results in inactivation of Gβγ subunits for α subunitbinding (Willard and Siderovski, 2006). It was found that small moleculeand peptide binding to the “hot spot” is retained. Some possible reasonsfor this are that primary amine coupling may affect binding of largermolecules to Gβγ but the binding site for small molecules remainsintact. Also, a dextran surface is not used herein for immobilizationbut rather the surface of the chip is coated with a PEG array (Lahiri etal., 1999). An advantage of this surface is the very low non-specificbinding. Additionally, because the density of reactive OH groups in thearray is lower than with dextran, the likelihood of immobilization bysingle linkages rather than multiple linkages is greater. As the numberof amines on the protein react with the surface the chances increase forinactivation of the protein. Regardless, the K_(d) obtained from theanalysis is consistent with what was obtained from other analyses.

To complement the SPR analysis, isothermal titration calorimetry (ITC)is used to determine binding constants. Advantages of this method overSPR include a lack of need for immobilization of the target molecule,the sensitivity is independent of the mass of the ligand, and additionalinformation about enthalpy can be obtained.

Expected results and interpretation: These analyses provide furtherevidence that the compounds are directly binding to Gβγ and give adirect determination of the affinity. Knowing the direct bindingaffinity gives a base from which to evaluate IC₅₀ values fromcompetition analysis between Gβγ subunits and effectors and allows forthe evaluation of the effects of Gβγ mutants on compound binding asdiscussed below.

(10) Mutagenesis of G Protein βγ Subunits and X-ray StructureDetermination to Define Compound Binding Modes

With direct binding assays for small molecule interactions with Gβγestablished, each selective compound can be investigated to determinethat a unique binding interaction determines effector selectivity.Computational modeling predicts unique binding interactions for manycompounds, but these models require validation.

(a) Mutagenesis:

A series of individual alanine substituted mutants of the G protein βsubunit were created and these mutants were used to determine the uniquebinding requirements for individual peptides that bind to the “hotspot”. Here, these mutants are screened for effects on binding to smallmolecules using the SPR assay. Individual purified mutants areimmobilized on biosensor chips and affinity constants calculated basedon k_(on) and k_(off) determined for binding of each compound andcompared with wt Gβγ. An initial focus is on M119 and M201 since thesehave such disparate effects on effector interactions. The predictedresult from this experiment is that distinct sets of amino acids arerequired for interaction of these molecules with Gβγ. Alaninesubstituted mutants for the majority of the amino acids in the “hotspot” have been created (Davis et al., 2005). These compounds bindwithin this region. These mutants have been analyzed extensively inother assays that confirm the functional folding and viability (αsubunit and peptide binding analysis) (Davis et al., 2005). M119 andM201 experiments establish evidence, but this analysis is performed onthe entire panel of binding compounds to date to create a binding mapfor each compound. Unique binding maps correlate with specific effectorspecificities.

(b) X-ray Crystallographic Determination of Ligand Binding to Gβγ.

The most definitive approach to defining the binding interactions ofthese small molecules with Gβγ is three dimensional structural analysiswith X-ray crystallography. This process is relatively straight-forwardbecause Gβγ has already been crystallized either alone or in complexeswith α subunits, effectors or peptides (Davis et al., 2005; Gaudet etal., 1996; Sondek et al., 1996; Wall et al., 1995). The focus is toobtain M119 or M201 Gβγ complexes. This is accomplished either byco-crystallizing Gβγ with compounds using conditions established forcrystallization of Gβγ alone (Sondek et al., 1996), or by screening newconditions, which are necessary when binding of the compounds affectscrystal lattice formation. A second approach is to soak preformedcrystals with compounds; this is tractable when binding sites are notalready occupied by crystal lattice contacts. The crystal lattice in thestructure of Gβ₁γ₁ and found relatively few crystal contacts with the“hot spot”. Additionally, there appeared to be extensive solventchannels in the lattice that allow diffusion of a small molecule in tothe “hot spot”. This indicates a crystal soaking approach is successful.

The determination of such structures is relatively straightforward usingeither difference Fourier methods or molecular replacement that employknown coordinates of Gβγ as a starting or search model. The initialco-crystallization efforts have focused on Gβγ with a γ₂C68S mutationand a 6His-tag at the amino terminus of γ₂. The γ₂C68S mutation preventspost-translational geranyl-geranylation of the γ subunit that targetsGβγ subunits to membranes. This soluble protein is easy to purify inrelatively high quantities (up to 10 mg/L) from the soluble fraction ofHi5 insect cell lysates using sequential Ni-NTA-agarose, HiTrap Q ionexchange and Superdex Gel filtration chromatography. The preparation ismonodisperse by dynamic light scattering. Hanging drop crystal screenswere based on previous communications with Dr Sprang and were based onconditions of 12-18% PEG 6K and 0.1M Tris-HCl pH 7-8.5 using proteinconcentrations between 2-8 mg/mL and conditions were expanded to includedifferent types of PEGs, as well as a broader range of proteinconcentrations, pH's and PEG percentages. Crystal formation was observedat a protein concentration of 5 mg/mL with pH 8.4 using 15% PEG 6K.Random screens were also setup to obtain possible alternate conditionsfor crystal formation and these were based on commercially availablecrystal screens from Qiagen®. In this screen a new crystal form wasidentified from 50% (v/v) PEG 200, 0.2 M NaCl buffered by 0.10 M Na+/K+phosphate pH 6.2. Screening is also underway to co-crystallize Gβ₁γ₂with compounds M119 and M201 using the conditions previously stated asoptimal for native Gβ₁γ₂(C68S) crystal growth. Alternativelygeranylgeranylated β₁γ₂ (with amino terminal 6His-tag on γ) purifiedfrom the membrane fraction of Hi5 cells can be utilized. The yield islower and the preparation more difficult but it is still a tractablesystem. 6His-tagged geranylgeranylated β₁γ₂ has been co-crystalized withthe SIGK peptide.

This analysis allowa definitive identification of the site ofinteraction of a given Gβγ binding molecule. Combined with SPR and ITCanalysis, a thorough understanding of the interaction is achieved.Individual compounds that have selective actions such as M201 and M119have distinct binding sites within the “hot spot” surface that can becorrelated with selectivity for different effectors.

(11) Computational Prediction of Gβγ Binding Specificity and Screeningof New Libraries.

Selectivity for specific Gβγ-target interactions is based ondifferential occupation of the “hot spot” surface. Thus, computationalscreening strategies can specifically target particular biologicalproblems. The current strategy is to screen the entire “hot spot” forbinders and to test these for competition with peptide followed bysystematic screening with several potential effector targets. Herein isdescribed potential “global analysis” approaches that streamlineevaluation of small molecules for selectivity. Because targeting asubsurface of the “hot spot” correlates with specific interrogation of aparticular pathway/biology, then a structurally defined subsurface (byX-ray crystallography or mutagenesis) can be targeted specifically forscreening with computational prediction methods. In broader terms,computational screens can be focused to target particular diseases byfocusing the screen to a discrete subsurface of the “hot spot”.

By developing a global screen for compound selectivity as describedherein, there is no need to target a specific “hot spot” subsite withcomputational screening. The “hot spot” is computationally screened aswas already done and use the global specificity screen to identifyselective binders. One issue is that the global specificity screen hasnot yet been developed. Secondly, by targetting a particular subsite apriori screening occurs more efficiently for the following reason: Inthe current screening strategy using any single scoring function is notthe most efficient at evaluating the quality of docked poses andpredicting high affinity binding of small molecules to the “hot spot”.Thus seven different scoring functions are used, each differing inapproach to evaluating the quality of the docked models. Differences inthe scoring function include whether the functions are based on trainingset of docked ligands or rely on first principles modeling of forcefield interactions (Brooijmans and Kuntz, 2003; Kitchen et al., 2004).In general, for a smaller binding site, one of these scoring functionstends to be the best at predicting true binders because a smaller sitehas a more limited set of interactions to be considered by theparticular scoring function. The reason that this is not true for the“hot spot” is that this large surface consists of many binding sites,each of which has unique physicochemical properties, so no one scoringfunction can predict the binding accurately. The logical extension ofthis argument is that focussing the screening to a unique binding siteas defined by X-ray crystallography or mutagenesis enables the use of asingle scoring function to identify binders for a particular “hot spot”subsurface. Using the current approach, compounds are tested that arehighly ranked in each of the seven scoring functions. When smalllibraries are screened such as the NCI diversity set (2000 compounds)this is not an issue, but when screening larger diversity sets, such asthe 50,000 compound diversity set from Chembridge, it is more of aproblem. For each computational screen, at least the top 1% of compoundspredicted by each scoring function are tested. For a 2000 compounddiversity set with 7 scoring functions this would be a maximum of 140molecules but for the 50,000 compound library this expands to 3500molecules. Use of a single scoring function reduces the number ofmolecules that would have to be tested by as much as seven-fold andreduces the screening to only 500 molecules.

Once a defined unique binding sites has been found, either by X-raycrystallography or mutagenesis, a computational screen is performedusing the Chembridge diversity set, focusing on one particular bindingsite identified for a selective PI3Kγ inhibitor. First the top 100compounds are screened from each scoring function (˜700 compounds) at aconcentration of 100 μM in the ELISA assay for inhibition of SIGKbinding and potential inhibitors are titrated to determine an IC₅₀. Thisnumber of compounds can be screened manually in a few weeks. When thecompounds bind to a single site it is understood that one particularscoring function will be particularly well suited to evaluating compoundbinding to that site and therefore one scoring function can predict ahigh proportion of compounds that bind with relatively high affinity.Binders that are identified are relatively selective for PI3Kγinhibition. The binders are tested for selective inhibition of PI3Kγ inassays that have been described in herein.

(12) Evaluation of Small Molecule Inhibitor Selectivity in a CellularModel of Inflammation.

The previous specific aims were designed to understand the mechanismsfor selectivity of Gβγ-binding compounds. One effector system ofparticular interest is PI3Kγ. PI3Kγ is relatively selectively expressedin neutrophils and several groups have shown that genetic deletion ofPI3Kγ results in impaired neutrophil migration in response tochemoattractants. Studies of neutrophil motility suggest that primaryevents that regulate directional neutrophil migration includeGβγ-dependent activation of PI3Kγ. These studies have attracted theattention of the pharmaceutical industry who have developed selectivecatalytic inhibitors of PI3Kγ relative to other PI3K isoforms as apotential anti-inflammatory strategy (Camps et al., 2005). Analternative approach to selective inhibition of PI3Kγ in neutrophilsthrough inhibition of PI3Kγ interactions with Gβγ is disclosed hereinsince the other isoforms of PI3K are primarily regulated by interactionwith tyrosine kinase receptors and/or ras, not through Gβγ. Indeed dataindicates that blocking Gβγ with an M119 related compound that blocksPI3Kγ regulation, (DL382), inhibits fMLP-dependent neutrophil chemotaxisand inhibits inflammation in an animal model.

Herein, potentially selective PI3Kγ inhibitors that have been identifiedusing the methods disclosed herein are tested. Data indicate that suchcompounds can be identified. For example, analysis indicates compound402959 (Table 18) binds to and inhibits peptide binding to Gβγ with anIC₅₀ of 2 μM, does not affect Gβγ-dependent regulation of PLCβ2 or β3but inhibits Gβγ-dependent PI3Kγ regulation with an IC₅₀ of 12 μM. Novelcompounds are identified that specifically inhibit Gβγ-dependentregulation of PI3Kγ, such as 402959, and test them in various assays ofneutrophil function in differentiated HL60 cells and primary humanneutrophils as described below. Compounds that affect other signalingprocesses such as GRK2 translocation or PLC activity but not PI3Kγ arealso tested. fMLP and IL-8 dependent regulation of PI3Kγ and PLC areexamined in neutrophils followed by assessment of neutrophil biologicalfunctions. Selective PI3Kγ inhibitors identified in vitro block PI3Kactivation in neutrophils and are effective blockers of chemotaxis butdo not affect Ca²⁺ signaling in neutrophils or neutrophil adhesion.Identification of compounds that block neutrophil chemotaxis but do notaffect Ca²⁺ signaling by fMLP are valuable anti-inflammatory inhibitorswith limited effects on other Gβγ-dependent signaling pathways.

(13) Intracellular Signal Transduction:

(a) PLC Activity.

PLC activity can be addressed by measuring receptor-dependent IP₃production and/or calcium efflux. Analysis of IP₃ production in thepresence and absence of compounds is conducted as described (Goubaeva etal., 2003). Briefly, differentiated HL60 cells are incubated overnightin inositol-free medium with 1 μCi [³H]inositol. Cultures arepreincubated with individual compounds for 5 minutes prior to additionof fMLP and incubated for 30 minutes at 37° C. IP₃ production isdetermined by scinitillation counting and reported as percent fMLPinduced IP₃.

Cytoplasmic calcium release is monitored in differentiated HL60 cellsand human neutrophils loaded with FURA-2 and resuspended in HEPESbuffered saline solution containing EGTA in the absence of extracellularCa²⁺ (as described in FIG. 5). M119 attenuates fMLP-induced Ca²⁺ releasein HL60 cells (FIG. 5A). Compounds that exhibit intrinsic fluorescenceare deemed unsuitable for this assay. Compounds not anticipated toaffect calcium levels or structurally similar compounds with lowaffinity for Gβγ are used as negative controls. To control for nonspecific effects on Ca²⁺ signaling compounds are also tested for effectson ionomycin-dependent Ca²⁺ release to ensure that this is not affected.

(b) PI3Kγ Activity.

PI3Kγ activity is assessed by evaluating translocation of GFP tagged Aktpleckstrin homology domain fusion protein (GFP-PHAkt) in stablytransfected HL60 cells (as described in FIG. 5 and (Servant et al.,2000)). Subcellular fractionation rather than microscopy has beenselected since it is a more objective evaluation. In agreement with invitro studies, M119 inhibited GFP-PHAkt membrane translocation in HL60cells (FIG. 5F). An alternative approach is to measure phospho-Akt witha specific phospho-Akt antibody.

(14) Neutrophil Function:

This subaim is designed to test the ability of compounds to modulateprocesses that are dependent on PI3Kγ and PLCβ2/3 activity in intactcells including superoxide production, chemotaxis and adhesion.

(a) Superoxide Production.

PLCβ2/3 and PI3Kγ have both been demonstrated to be involved insuperoxide production (Li et al., 2000). Therefore, compounds thatinhibit either or both PLCβ2 and PI3Kγ are screened for effects onsuperoxide production in HL60 cells and human neutrophils. Superoxideproduction is measured as described (Vlahos et al., 1995). Briefly,cells are plated in a 96-well plate in HEPES buffered saline solutioncontaining cytochrome c to which compounds/DMSO or PMA (positive control(Li et al., 2000)) is added. After a 5 minute preincubation, fMLP isadded and incubated for 5 minutes. Superoxide production is reported assuperoxide (nM/min) as determined by absorbance at 550 nm over a 5minute time period. Compounds that inhibit both PLCβ and PI3Kγ are morepotent than compounds that inhibit one or the other selectively and thatthese compounds do affect PMA stimulated superoxide production.

(b) Chemotaxis.

Given the defined role of PI3Kγ in neutrophil chemotaxis (Curnock etal., 2002; Li et al., 2000; Sasaki et al., 2000), compounds that inhibitin vitro PI3Kγ activity are evaluated for effects on fMLP-inducedchemotaxis in the Boyden chamber as described (Hannigan et al., 2002),and in data FIGS. 10E and F. Wortmannin is used as a positive controlfor inhibition of chemotaxis. It is important to note that certaincompounds may prevent neutrophil chemotaxis (directed migration in agradient of stimulus), but the neutrophils may still undergochemokinesis (random, stimulus-dependent migration). Recent literaturedemonstrated that PI3Kγ deficient neutrophils are unable to translocateup the chemotactic gradient, but still undergo chemokinesis (Li et al.,2000). Therefore, to control for chemokinesis fMLP is added to bothchambers and cells that translocate through the membrane are consideredchemokinetic.

(c) Cell Adhesion.

In these assays, neutrophil adhesion to extracellular matrix is measuredin response to chemokine stimulation. In these assays, endothelialligands are presented in conjunction with immobilized chemokines (IL-8).In the absence of IL-8, no adhesion is observed indicting thatactivation of the GPCR dependent pathway known to involve Gi and Gβγ isrequired for the observed adhesion. Results indicate that wortmannindoes not block this process indicating that pathways other than PI3Kγactivation are responsible for adhesion. This allows for testing theselective compounds to determine influence on adhesion or chemotaxis.

Specific adhesion method: A dual micropipette micromanipulation systemis used to measure adhesion probability between cells (neutrophils,principally) and target beads coated with specified endothelial ligands(typicaly ICAM-1) (Lomakina and Waugh, 2004; Lomakina and Waugh, 2006).One pipette is used to hold the target bead, and one is used to hold thecell, and the operator manually brings the cell and bead into repeatedcontact. Detection of adhesive events depends on visual observation ofthe cell surface as the cell and bead are separated. An adhesive eventis detected as a small deformation of the cell surface duringseparation. The probability of adhesion is simply the number of adhesivecontacts out of the total number of contacts between the cell and bead.Typically 25 contacts are used for an individual determination.Alternatively, adhesion probability can be monitored continuously toobserve the time course of the change in cell adhesiveness afterstimulation.

Compounds that identified that are selective for blocking Gβγ-dependentPI3Kγ activation or with other selectivity have similar selectivityprofiles for signal transduction pathways in intact cells and block theexpected physiological response of the neutrophil. For example, aninhibitor that selectively blocks Gβγ-dependent regulation of PI3Kγblocks chemotaxis but does not block Ca²⁺ signaling or IL-8 dependentneutrophil adhesion. Compounds that block GRK2 regulation or PLCβ do notto affect chemotaxis but other processes such as neutrophil adhesion orsuperoxide production are affected. In order to be considered aselective regulator of intracellular signaling, the compound must beshown to inhibit some intracellular function to be sure that thecompound is indeed cell permeable and effective.

(15) Compounds and Libraries:

All of the compounds that were used to generate data and are used in theexperiments described herein have been obtained through theDevelopmental Therapeutics Program at the National Cancer Institute. TheNCI diversity set library contains 1990 compounds in 200 μl DMSO at 10mM in a 96 well format. The diversity set was assembled to representchemical diversity in the larger 250,000 compound NCI library. As leadcompounds are identified from screening the diversity set, structurallyrelated compounds identified by similarity searching were obtained incrystalline form for all of the lead compounds in Table 18 in 10 mgquantities which is more than sufficient for all of the proposedexperiments. These compounds have been freely available in the past, butmore recently have become freely available only if a cancer relatedjustification can be provided. Since G protein signaling has roles inmitogenesis and cancer cell migration, this justification has not beendifficult. Additionally, some of the compounds such as M201 and DL382are available in high purity and quantity from commercial vendors. Apotential issue with many of the compounds in the diversity set is thelevel of purity of the compounds. In all cases, the biological efficacyand potency of identified compounds have been confirmed with freshpreparation from the crystalline form and in some cases with furtherconfirmation of efficacy and potency with pure preparations from othersources. Compounds that have not been analyzed for purity are analyzedfor purity and identity by HPLC and GC-MS analysis. Another issue isthat many of the compounds in this library contain metals that are redoxactive might give false positive results. For all compounds screenedoxidation dependent activity was assayed for by adding DTT to the assayand metal ion dependent activity using metal chelators. Compounds thatappear to work through these mechanisms are not considered further.

It is contemplated herein that other libraries such as the Chembridge50,000 compound DIVERSet collection can be screened. This set representsthe compounds in a larger 435,000 compound express-PICK library.

(16) Human Neutrophil Preparation.

Whenever possible, neutrophils are selected directly from whole bloodsamples dispersed into low endotoxin buffers. They are easily identifiedby their size and multilobular nuclear structures. When necessary,purified neutrophil populations are isolated from whole blood by densityseparation. Venous blood drawn from healthy donors is placed over alayer of 1-Step Polymorphs (Accurate Chemical & Scientific Corporation,Westbury, N.Y.). After centrifugation at 1500 rpm for 45 minutes, theband of polymorphonuclear cells is visible. Neutrophils are harvested bypipette, then washed in 4% FCS in Hanks Balanced Salt Solution (HBSS,BioWhittaker, Walkersville, Md.), containing 10 mMN-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES, Sigma,Saint Louis, Mo.) without Ca²⁺ and Mg²⁺, and brought to finalconcentration 5×10⁶ cells/ml. Measurements are completed within sixhours after phlebotomy.

(17) Fluorescence Screening for Gbg “hot spot” Molecules

Two new assays have been developed that allow more effective screeningfor Gβγ “hot spot” molecules. Both involve using a fluorescently labeledversion of SIGK with fluorescein attached at the amino terminus. Thefirst is a fluorescence polarization anisotropy (FP) assay where bindingof Gβγ to the peptide increases the anisotropy of the fluorescence dueto rotational slowing of peptide motions. The second is a fluorescencequenching method where binding of the peptide to Gβγ quenches peptidefluorescence by an unknown method. Both methods are highly amenable toHTS screening in a homogeneous single addition assay format where theassay would screen for compounds that block Gβγ peptide interactions byassaying for compound ability to reduce anisotropy or fluorescencequenching.

i) Materials and Methods

(1) Small Molecules.

Compounds for this study were kindly provided by the National CancerInstitute repository except when indicated otherwise. Compoundabbreviations are as follows: M119 (NSC119910) and M119B (NSC119892).Gallein (Acros Organics, Geel, Belgium), fluorescein, and wortmanninwere obtained from Sigma Aldrich (St. Louis, Mo.).

(2) Competition ELISA and Structure Activity Relationships.

Binding of small molecules to Gβ₁γ₂ was assessed by competition withphage displaying the SIGK peptide as described previously (Scott et al.,2001

; Bonacci et al., 2006). In brief, Gβ₁γ₂ (25 nM), with biotinincorporated via an N-terminal acceptor peptide on Gβ₁, was immobilizedin a 96-well plate coated with streptavidin. Compounds/DMSO and 0.1×10¹⁰phage were added simultaneously and subsequently incubated for 1 h atroom temperature. Plates were then washed with 1× Tris-bufferedsaline/0.5% Tween 20 and incubated with anti-M13, HRP-conjugatedantibody (Amersham, Chalfont St. Giles, Buckinghamshire, UK). Phagebinding was determined by monitoring A₄₀₅ upon addition of2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (Sigma Aldrich).

(3) Surface Plasmon Resonance.

Direct binding of “hot spot” binding small molecules was assessed usingthe Reichert SR7000 Surface Plasmon Resonance dual chamber instrumentequipped with an autosampler (Reichert, Depew, N.Y.). To activate thesensor chip surface, a mixture of 0.1 M1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and 0.05 MN-hydroxysuccinimide was injected (flow rate 5 μl/min) over the sensorsurface. Streptavidin (100 μg/ml; prepared in 20 mM sodium acetatebuffer, pH 5.5) was coupled to the sensor chip surface followed byquenching the remaining activated carboxyl groups with 1 M ethanolamine,pH 8.5. bGβ₁γ₂ was conjugated to the streptavidin-coated chip in runningbuffer (50 mM HEPES, pH 7.6, 1 mM EDTA, 100 mM NaCl, 0.1% C₁₂E₁₀, and 1mM dithiothreitol) to achieve 2000 micro-refractive index units. Asecond reference cell was treated similarly, but bGβ₁γ₂ was excluded.Direct binding of small molecules was tested out at room temperaturewith a flow rate of 75 μl/min. Compounds were prepared in running bufferand injected for 10 min followed by a dissociation phase of 10 min. Theobtained sensorgrams were corrected for nonspecific background byrunning the same experiment in series over an identical sensor surfacewith immobilized streptavidin, blocked with biotin, without bGβγ.Binding rates and constants were independent of flow rate over a widerange and did not fit mass transport limited models, indicating thatmass transport was not limiting association or dissociation rates. Bestfit kinetic parameters were determined using kinetic titration model andClampXP ver. 3.5, which accounts for incomplete dissociation betweeninjections (Karisson et al., 2006).

(4) GFP-PH-Akt Translocation in Differentiated HL60 Cells.

HL60 cells stably overexpressing GFP-PH-Akt (Servant et al., 2000) weremaintained and differentiated as described previously (Bonacci et al.,2006). Cells (0.2×10⁶ cells/ml) were differentiated by incubation with1.2% DMSO for 5 days. Cells were washed with serum-free RPMI 1640(Invitrogen, Carlsbad, Calif.). Cells (2×10⁵; 100 μl) were transferredto a 1.8-ml Beckman ultracentrifuge tube. Cells were pretreated withDMSO or compounds for 10 min at room temperature and stimulated with 250nM fMLP (Sigma Aldrich) for 2 min. at 37° C., snap-frozen in liquidnitrogen, and thawed in the presence of 100 μl of 2× lysis buffer (100mM HEPES, pH 8.0, 6 mM MgCl₂, 0.2 mM EDTA, 200 mM NaCl, 100 μMNa₃VO₄,and protease inhibitors). Membranes were harvested by centrifugation100,000 g for 20 min after four freeze-thaw cycles. All treatmentscontained the same final concentration of DMSO. Pellets were washed oncewith 100 μl of lysis buffer (50 mM HEPES, pH 8.0, 3 mM MgCl₂, 0.1 mMEDTA, 100 mM NaCl, 50 μM Na₃VO₄, and protease inhibitors), pelleted asabove, and boiled in 2× sample loading buffer. Samples were resolved by12% SDS-PAGE, transferred to nitrocellulose, and probed with anti-GFPantibody (1:1000; Roche) followed by incubation with goat anti-mouseHRP-conjugated secondary antibody (1:5000, Bio-Rad Laboratories,Hercules, Calif.). Chemiluminescence was analyzed using a charge-coupleddevice camera in a UVP Epi-Chem II Darkroom imaging system. All sampleshad background subtracted and were normalized to the fMLP-induced signalset at 100%.

(5) Cell Motility Analysis.

Chemotaxis was assayed using a Boyden chamber (Neuro Probe,Gaithersburg, Md.) using 3-μm polyvinylpyrrolidone-free polycarbonatefilters (Neuro Probe). HL60 cells (differentiated for 4 days) werewashed with and resuspended in HBSS containing 1% bovine serum albuminto a final concentration of 1×10⁶ cells/ml. Primary human neutrophilswere washed and resuspended in NaCl buffer (140 mM NaCl, 4 mM KCl, 10 mMD-glucose, 10 mM HEPES, pH 7.4, 1 mM MgCl₂, and 1 mM CaCl₂) to a finalconcentration of 1×10⁶ cells/mi. Chemoattractant [1 μMgranulocyte-macrophage-colony stimulating factor (GM-CSF), 10 nM IL-8,or 250 nM fMLP; Sigma Aldrich] was added to the bottom chamber in HBSScontaining 1% bovine serum albumin. Cell suspensions (0.2×10⁶cells/well) were added to the top wells of the Boyden chamber andallowed to migrate for 1 h at 37° C. When applicable, cell suspensionswere preincubated for 10 mM with small-molecule inhibitors (in DMSO) atthe indicated concentrations, and the bottom chamber was adjusted to thesame concentration of small molecule. All treatments contained the samefinal concentration of DMSO. Filters were processed according to themanufacture's recommendations and stained using DifQuik (VWR Scientific,West Chester, Pa.). Chemotactic HL60 cells were scored by counting threemicroscope fields and subtracting the number of cells from fMLP wells asbackground. All samples had background subtracted and were normalized tothe fMLP-induced signal set at 100%. For all the small molecules,effects of the compounds on chemokinesis was analyzed by measuringchemotaxis in the presence of 250 nM fMLP in both the upper and lowerchambers of the Boyden Chamber and measuring changes in transwellmigration. Unless otherwise indicated, none of the compounds had effectson chemokinesis.

(6) Measurement of Superoxide Production.

The nitro blue tetrazolium (NBT) method was used to assess the effectsof Gβγ inhibitors on NADPH oxidase activity. HL60 cells (differentiatedfor 4 days) were washed with and resuspended in HBSS containing calciumand magnesium (Cellgro; Mediatech, Herndon Va.) to a final concentrationof 2×10⁶ cells/ml. Cells (1×10⁶/reaction) were pretreated with 10 μMGβγinhibitor compound (in DMSO) or 100 nM wortmannin (Sigma Aldrich) for 10min at 37° C. before the addition of NBT (25 μl of 10 mg/ml in methanol)and then incubated or an additional 5 min at 37° C. All treatmentscontained the same final concentration of DMSO. Cells were thenactivated with either 250 nM fMLP (Sigma Aldrich) or PMA (Sigma Aldrich)for 30 min in a 37° C. water bath. Reactions were stopped by theaddition of 500 μl of 1.2 N HCl, and cells were collected bycentrifugation at 12,000 g for 5 min. Cell pellets were then resuspendedin 200 μl of DMSO, transferred to a 96-well plate, and absorbance wasmeasured at 540 nM. All treatments and controls contained the sameconcentration of DMSO. All samples had background subtracted and werenormalized to the fMLP-induced signal set at 100%.

(7) Evaluation of Rac-1 Activation.

HL60 cells (differentiated for 4 days) were washed with and resuspendedin NaCl buffer to a final concentration of 20×10⁶ cells/ml. Cells(10×10⁶/reaction) were pretreated with 10 μMGβγ inhibitor (in DMSO) for10 min before challenge with 1 μM fMLP for 90 s in a 37° C. water bathand then immediately transferred to an ice-water bath. Cells wererecovered by centrifugation at 500 g and washed two times with ice-coldTris-buffered saline, pH 7.4. The Rac1 activation assay kit (UpstateCell Signaling Solutions, Billerica, Mass.) was used to prepare cellextracts and evaluate Rac1 activation according to the manufacturer'sinstructions. Affinity-purified GTP-Rac1 was resolved by 15%SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose,probed with anti-Rac1 monoclonal antibody followed by detection withHRP-conjugated secondary antibody in accordance with the manufacturer'srecommendations. Chemiluminescence was analyzed using a charge-coupleddevice camera in a UVP Epi-Chem II Darkroom imaging system. Results wereexpressed as percent fMLP-stimulated GTP Rac1 normalized to total Rac1with the background subtracted.

(8) Carrageenan-Induced Paw Edema and Neutrophil Abundance.

Male Swiss-Webster mice (35-40 g b.wt.; Taconic Farms, Germantown, N.Y.)were randomized and acclimated for 1 week in a 12-h light/dark cycle.Food and water were provided ad libitum. Animals were labeled withunique identifiers on their tails using indelible marker. One hourbefore challenge with carrageenan, mice were administered compounds(dissolved in PBS) or vehicle (PBS) either by intraperitoneal (300 μlwith 27.5 gauge needle) injection or by oral gavage (100 μl with 1.5″,steel ball-tipped feeding needle; Popper and Sons, New Hyde Park, N.Y.).Indomethacin (2.5 mg/kg) stock solution was prepared in methanol anddiluted into PBS (Mediatech), small-molecule inhibitor (concentration asindicated in appropriate figure legend) was prepared in PBS. Mice wereanesthetized by intraperitoneal injection with ketamine hydrochloride(175 mg/kg) and xylazine (7 mg/kg) using a 26 gauge needle. Mice weretested for pain reflex to ensure sedation and then injectedsubcutaneously into the plantar region of the hind paw with 25 μl of 2%carrageenan using a precision engineered ⅝″ 25 gauge Hamilton syringe(hypodermic needle; Hamilton Co., Reno, Nev.). Carrageenan (CarboMer,Inc., San Diego, Calif.) was suspended in PBS at 50° C. for 10 min withstirring the night before experimentation. The contralateral paw wasinjected with vehicle as control. Mice were then transferred to beddedcages to minimize pain associated with the carrageenan injections.Dorsal-plantar swelling was measured using an electronic digital caliper(±0.03 mm; VWR Scientific) at time 0 and every 2 h thereafter. To ensuremeasuring and injection consistency, the dorsal and plantar surface ofthe test paw and contralateral paws of each animal were marked withindelible marker. Each paw was measured two times at each time point andaveraged. At the conclusion of the experiment, animals were euthanizedin accordance with the University of Rochester and American VeterinaryMedical Association standards by carbon dioxide narcosis and cervicaldislocation. Paw edema was determined by subtracting the thickness ofthe contralateral paw from that of the carrageenan-injected paw at eachtime point.

Paws were amputated 2 h after carrageenan injection to determine thenumber of neutrophils present in the edematous fluid. Paws weretransferred to prepared Eppendorf tubes, and the exudates were collectedby centrifugation for 2 min. The paws were removed and the tubesre-weighed to determine the mass of the exudates, which was thenconverted to volume. The volume in the contralateral untreated controlpaw was subtracted from the cardgeenan-treated paw volume. To removeerythrocytes by hypertonic lysis, the exudate was resuspended in 250 μlof 1× PBS to which 50 μl of water was added. At the conclusion of a 30-sincubation, 75 μl of 4.5× PBS was added to return the solution to normalsalt levels. The number of neutrophils present was determined by manualcounting, and the small number of neutrophils in the controlcontralateral paw was subtracted.

(9) Isolation of Primary Human Neutrophils.

Human blood obtained by venous puncture from consenting, healthy adultdonors, in accordance with University of Rochester standards, andcollected in sterile vacutubes containing sodium heparin (BDBiosciences, San Jose, Calif.). Neutrophils were layered overPolymorphprepT11 (Accurate Chemical and Scientific Co., Westbury, N.Y.)and isolated by centrifugation (470 g for 50 mM at room temperature).Trace erythrocytes were removed by hypertonic treatment followed bycentrifugation. Isolated neutrophils were stored in HEPES-bufferedsaline solution (146 mM NaCl, 5 mM KCl, 5.5 mM D-glucose, 10 mM HEPES, 1μM CaCl₂, and 1 mM MgSO₄) at pH 7.4.

(10) Data Analysis.

Ligand competition curves were determined by nonlinear regression usingPrism software (GraphPad Software, Inc., San Diego, Calif.). Statisticalsignificance was evaluated by one-way analysis of variance andBonferroni's multiple comparison test. Statistical significance wasdefined as *, P<0.05; **, P<0.01; ***, P<0.001.

3. Example 3 A Novel Gβγ-Subunit Inhibitor Selectively Modulates Acuteμ-Opioid-Dependent Antinociception and Attenuates Morphine-InducedAntinociceptive Tolerance and Dependence

Opioid analgesics, such as morphine, are clinically important for thetreatment of moderate to severe pain. Molecular cloning experiments ledto the identification of the mu (μ), delta (δ), and kappa (κ) opioidreceptors (Evans et al., 1992; Kieffer et al., 1992; Chen et al., 1993).All three types of opioid receptor are GPCRs (G protein coupledreceptors) that activate G_(i) resulting in inhibition of adenylylcyclase activity (Childers, 1991; Sharma et al., 1977), activation ofinwardly rectifying K⁺ channels (Armstrong et al., 1992; North et al.,1987), inhibition of voltage-activated Ca²⁺ channels, (Moises et al.,1994; Schroeder et al., 1991), the activation of the MAP kinase (mitogenactivated protein kinase), Erk-1/2 (extracellular signal-regulatedkinases) (Law et al, 2004) and phosphatidylinositol (PI) specificphospholipase C (PLC) (PI-PLC) (Xie et al., 1999). Recently, theimportance of Gβγ-mediated activation of PI-PLC in the potentiation ofopioid analgesia has been examined (Xie et al., 1999, Galeotti et al.,2006).

Activation of PLC results in hydrolysis of phosphatydilinositol4,5-bisphosphate (PIP₂) to inositol 1,4,5, triphosphate (IP₃), whichmobilizes Ca²⁺ from intracellular stores, and diacylglycerol (DAG),which activates protein kinase C (PKC) (Rebecchi et al., 2000). PLCβ2and PLCβ3 isoforms are activated by Gβγ and are responsible for PIhydrolysis stimulated by G_(i) coupled receptors, with PLCβ2 beingexpressed primarily in hematopoetic cells (Rhee et al., 1997).

Data indicated that pharmacological inhibition of PLCβ3 enhancedopioid-induced antinociception. In these experiments with PLCβ3knock-out mice, deletion of PLCβ3 resulted in a 10-fold potentiation inanalgesic response in mice treated with morphine, compared to controlanimals, in the 55° C. warm-water tail-flick assay. This was one of thefirst indications that this pathway is an important regulator of opioidsignaling and subsequent analgesic responsiveness, and indicated thattargeting PLCβ3 or PLCβ3 regulation pharmacologically influenced opioidefficacy.

From screening of a small molecule library (NCI diversity set) severalcompounds were found that bound to Gβγ subunits and selectivelyinhibited Gβγ subunit signaling in vitro (Table 20). The lead compoundin the series, M119, had high affinity for the Gβγ subunit and was aninhibitor of PLCβ3 signaling in vitro. In vivo, co-administration ofM119 (100 nmol, i.c.v.) with graded doses of morphine (i.c.v.) resultedin a 10-fold leftward shift in the morphine dose-response curve(Bonacci, et al., 2006). This same shift is observed in PLCβ3 knock-outmice that have been treated with morphine alone (Bonacci et al., 2006;Xie et al., 1999). Administration of M119 with morphine in the PLCβ3knock-out mice had no additional effect (Bonacci et al., 2006), furthersupporting the hypothesis that the mechanism of action for M119 wasthrough the attenuation of opioid-induced activation of PLCβ3 by Gβγ. Itis important to note that morphine still produced an analgesic responsein the animals which had been administered M119, indicating thatregulation of other Gβγ targets was still intact.

TABLE 20 Structure-Activity Relationship (SAR) of M119 based oncompetition ELISA analysis for SIGK binding. Compound* IC₅₀ ELISA M119(119910) 0.2 μM M119B (119892) >300 μM M119C (119911) 0.2 μM M119D(119912) 30 μM M119E (119913) 0.7 μM M19F (119915) >300 μM M119G (11916)5 μM M119H (119888) >300 μM M119I (119891) NB M119J (119894) >300 μMM119K (119893) 0.13 μM M122 (122390) 14 μM M157 (157411) 70 μM M158(158109) NB M158B (158113) NB M158C (158110) 0.25 μM M158D (158112) >300μM M260 (2608) 300 μM M542 (5426) NB M903 (9037) >300 μM *NCIidentification numbers in parentheses. NB- No inhibition at 300 μM. Dataare from Bonacci et al. (2006).

Selectively inhibiting downstream signaling from the Gβγ subunit, with asmall molecule inhibitor, is a novel approach to targeting only apathway of interest, while leaving the rest of the signaling machineryintact. To take this concept a step further, the goal of this currentstudy was to determine the effect M119 would have in vivo, not only onantinociception mediated by all three opioid receptors, but also inmodels of acute analgesic tolerance and dependence.

a) Materials and Methods

(1) Animals

Male, ICR mice (20-30 g) (Harlan Industries, Indianapolis, Ind.) werehoused in groups of five with food and water available ad libitum beforeany procedures. Animals were maintained on a 12-hr light/dark cycle in atemperature-controlled animal colony. Studies were carried out inaccordance with the Policies on the Use of Animals in NeuroscienceResearch.

(2) Chemicals

M119 (cyclohexanecarboxylic acid,2-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)-(9cl)) (FIG. 3C) was obtainedfrom the chemical diversity set from the Developmental TherapeuticsProgram from the NCI/NIH. M119 is compound 119910 within that series.Morphine sulfate was purchased from Mallinckrodt (Saint Louis, Mo.).[D-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin (DAMGO),[D-Pen²,D-Pen⁵]enkephalin (DPDPE), [D-Ala²]-Deltorphin II (DeltorphinII),(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamidemethane-sulfonate hydrate (U50,488), β-FNA and naloxone were purchasedfrom Sigma Chemical Co. (Saint Louis, Mo.). Scintisafe was purchasedfrom Fisher Scientific (Pittsburgh, Pa.).

(3) Inositol Phosphate Assay Using hMOR-CHO, hKOR-CHO, and hDOR-CHOCells.

Chinese hamster ovary (CHO) cells stably expressing either the human κ(hKOR-CHO), δ (hDOR-CHO)(L. Toll, Stanford Research Institute, PaloAlto, Calif.) or μ (hMOR-CHO)(G. Uhl, National Institute on Drug Abuse,Baltimore, Md.) opioid receptor were used in the experiments. Cells in6-well plates were labeled by adding 4 μCi of [³H]inositol for 24 hr ininositol-free F-10 media, without serum. After labeling, LiCl was addeddirectly to the labeling media at a final concentration of 10 mM.Ligands or peptides were added at the same time. The final volume ofeach well was 1 mL. The plates were put back in to the incubator at 37°C. for 30 min after which time the medium was aspirated and the plateswere washed twice with 1× PBS. Ice-cold 50 mM formic acid, 1 mL, wasadded to the plates which were placed in the cold room at 4° C. for 30min. After the incubation, the contents of the plates were applied toDowex AG1-X8 columns and allowed to flow all the way through the column.The columns were washed, twice each, with 50 and 100 mM formic acid, fora total of four washes, followed by elution of the IP-containingfraction with 3 mL of 1.2 M ammonium formate/0.1 M formic acid. Theeluted fraction was mixed with Scintisafe scintillation fluid, for highsalt, and counted. The data are represented as the fold increase overcontrol in total inositol phospates. All experiments were repeated threetimes and performed in duplicate. Control cells for each experiment werelabeled with [³H]inositol and LiCl, in the same manner as the treatmentgroups.

(4) Drug Solutions and Injections

For intracerebroventricular (i.c.v.) injections, M119 was initiallysolubilized in DMSO and all subsequent dilutions were in distilledwater. For systemic injections intraperitoneal (i.p.), M119 wasinitially solubilized in a small volume of NaOH and brought to volume inphosphate buffered saline (PBS) to approximately pH 7.5. Vehicle wasprepared in a similar manner without the drug. Additional compounds usedin these studies were dissolved in the same vehicle as M119 for bothi.c.v. and systemic injections. Intracerebroventricular injections weremade directly into the lateral ventricle according to the modifiedmethod of Haley and McCormick (1957). The mouse was lightly anesthetizedwith ether, an incision was made in the scalp, and the injection wasmade 2 mm lateral and 2 mm caudal to bregma at a depth of 3 mm using a10-μl Hamilton syringe. The volume of all i.c.v. injections was 5 μl.

(5) Antinociceptive Testing

Antinociception was assessed using the 55° C. warm-water tail-flicktest. For the tail-flick test, the latency to the first sign of a rapidtail-flick was taken as the behavioral endpoint (Jannsen et al., 1963).Each mouse was first tested for baseline latency by immersing its tailin the water and recording the time to response. Mice not respondingwithin 5 sec were excluded from further testing. Mice were thenadministered the test compound and tested for antinociception 20 minafter the injection. A maximum score was assigned (100%) to animals notresponding within 15 sec to avoid tissue damage. Antinociception wascalculated by the following formula: % antinociception=100×(testlatency−control latency)/(15−control latency).

(6) Effects of M119+ Receptor-Selective Agonists

Mice were treated concomitantly with M119 (100 nmol, i.c.v.) and gradeddoses of receptor selective agonists (DAMGO, U50,488, DPDPE, andDeltorphin II). Control mice received a vehicle injection (i.c.v., −20min). Antinociception was assessed 20 min after agonist injection. Totest the effects of M119 systemically, mice received M119 (100 mg/kg,i.p.) followed immediately by graded doses of morphine subcutaneous(s.c.). Antinociception was tested 20 min following the agonistinjection.

(7) Acute Antinociceptive Tolerance

For quantitative measurements of acute opioid tolerance, a standardizedstate of tolerance was induced by administration of morphine at times 0,2 hr, 4 hr, and 6 hr. The degree of tolerance was calculated from theshift in ED₅₀ value from the non-tolerant state to the tolerantcondition (Way et al., 1969). All injections were i.c.v. andantinociception was assessed 20 min after each injection. Mice werelightly anesthetized prior to each injection. Previous reports haveindicated that this dosing schedule induced acute morphine tolerance(Jiang et al., 1995). To assess the role of M119 in acute morphinetolerance M119 and morphine were administered concomitantly at times 0,2 hr, 4 hr, and 6 hr. Time 0 is defined as the first injection ofagonist.

(8) Acute Physical Dependence

To assess development of acute morphine physical dependence (Yano andTakemori, 1977; Bilsky et al., 1996, Wang et al., 1999) mice werepretreated with a single injection of morphine (100 mg/kg, s.c., −4 hr).Prior to the administration of naloxone (30 min) mice were injected withvehicle, i.c.v. Withdrawal was precipitated by an injection of theopioid antagonist, naloxone (10 mg/kg, i.p.). Mice were immediatelyplaced in a clear cylinder and observed for 15 min. The number ofvertical jumps was recorded during this time. To test if M119 affectedmorphine physical dependence, the same treatment as described above wasused with M119 (100 nmol, i.c.v.) being administered 30 min prior to theadministration of naloxone.

(9) Statistical Analysis

Data from dose-response experiments were fitted to a sigmoidaldose-response model using nonlinear regression analysis, and ED₅₀ valueswere calculated. A shift in the dose-response curve was determined fromthe ED₅₀ values. To assess if the ED₅₀ values for 2 dose-response curveswere significantly different, an F-test was performed (GraphPad Prims4.03). Values are reported as two-tailed p values. Statisticalsignificance was set at P<0.05. All data points are the mean of 7-10mice, with standard error of the mean represented by error bars.Statistical analysis of the acute dependence data and inositol phosphatedata used the Student's t test.

b) RESULTS

(1) The Gβγ Inhibitor, M119, Potentiated μ-Mediated Antinociception.

The efficacy of the compounds were tested in an in vivo model. Comparedto wild type animals, PLCβ3 −/− mice are 10-fold more sensitive to theantinociceptive effects of the μ-agonist morphine (Xie et al., 1999).Since M119 blocks Gβγ-dependent activation of PLCβ3 it was determinedwhether co-administration of M119 with morphine would also increasemorphine-induced antinociception. Co-administration of M119 withmorphine intracerebroventricularly (i.c.v) resulted in an 11-foldincrease in the analgesic potency of morphine (FIG. 14A) [ED₅₀ valuesand 95% confidence limits: 0.069 (0.023-0.201) nmol and 0.743(0.341-1.62) nmol respectively], whereas administration of 100 nmol M119alone had no effect on baseline antinociception. M119 also had no effecton morphine-dependent antinociception in PLCβ3 −/− mice (FIG. 14B).These data highlight the specificity of M119 actions and the selectivenature of M119 both in vitro and in vivo. Gβγ subunits regulate manyaspects of signaling critical for the actions of opioid agonists (Connorand Christie, 1999). If M119 were globally blocking Gβγ subunitfunctions, it is expected that morphine-induced antinociception wouldhave been attenuated rather than potentiated with M119co-administration.

Data disclosed herein demonstrated that PLCβ3, a Gβγ subunit-regulatedenzyme, was a negative modulator of μ opioid-dependent signaling.Therefore, it was predicted that inhibiting βγ-dependent PLCβ3activation would potentiate μ opioid-mediated antinociception, in amanner similar to the PLCβ3 knock-out studies (Xie et al., 1999, Bonacciet al., 2006). Additionally, it was observed that co-administration ofM119 (FIG. 3C) with morphine resulted in a significant (p<0.001),10-fold potentiation in morphine-mediated antinociception (Bonacci etal., 2006) (FIG. 14). To determine if the effects of M119 were specificto morphine, M119 was also administered with agonists selective for theμ, κ, and δ receptors. Co-administration of M119 and the μ-selectiveagonist, DAMGO, resulted in a 7-fold leftward shift in the DAMGOdose-response curve when compared to DAMGO alone (p<0.001). DAMGO aloneproduced an ED₅₀ value and 95% CL of 0.07 nmol (0.03-0.17 nmol) whileDAMGO and M119 resulted in an ED₅₀ value and 95% CL of 0.01 nmol(0.005-0.03 nmol). A modest, 2-fold, shift was observed in thedose-response curve of the κ-selective agonist, U50,488, whenadministered with M119 (p<0.01). The ED₅₀ values for U50,488 alone andU50,488 with M119 were 37 nmol (29-47 nmol) and 17 nmol (9.4-31) nmol,respectively. M119 did not potentiate antinociception mediated througheither δ₁ or δ₂ receptor. The ED₅₀ values for the δ₁-specific agonist,DPDPE, alone or with M119 did not change significantly, 6.5 nmol (2.6-16nmol) and 5.4 nmol (2.6-11 nmol), respectively. The δ₂-selectiveagonist, Deltorphin II, produced an ED₅₀ value of 11 nmol (5.5-23 nmol)which was unchanged in the presence of M119, 11 nmol (5.1-23 nmol).Administration of M119 alone had no effect on baseline tail-withdrawallatancies (Bonacci et al., 2006). These experiments demonstrated that,in vivo, M119 selectively potentiated μ opioid-dependentantinociception.

To determine if M119 would be effective after systemic administration,M119 was administered i.p. followed immediately with a s.c. injection ofmorphine. Systemic administration of morphine alone produced an ED₅₀value of 5.0 mg/kg (2.9-8.4 mg/kg) which was shifted 4-fold to the leftin the presence of M119, producing an ED₅₀ value of 1.3 mg/kg (0.59-2.8mg/kg) (p<0.001).

(2) M119 Selectively Inhibited Inositol Phosphate Generation MediatedThrough The IA Receptor.

It is disclosed herein that the potentiation in morphine-inducedantinociception was due to M119 inhibition of βγ-dependent PLCβ3. Todemonstrate that M119 blocked μ-opioid-receptor-dependent PLCactivation, morphine and DAMGO-dependent total inositol phosphate (IP)production were measured in hMOR-CHO cells. Both DAMGO (10 μM) andmorphine (10 μM) significantly increased total IP measured as comparedto control, with a 4- and 3-fold increase respectively (p<0.01) (FIG.15A). This increase was significantly attenuated with the addition ofthe βγ-inhibitor, M119 (10 μM) (p<0.05) while M119 had no effect byitself. The μ-receptor selective antagonist, β-FNA (β-funaltrexamine),attenuated DAMGO and morphine-dependent IP production (FIG. 15A)(p<0.05). The κ-selective agonist, U50,488 (10 μM) and the δ-selectiveagonist, DPDPE (10 μM) had no effect on IP production alone or in thepresence of M119 in the hMOR-CHO cells (data not shown). These datademonstrate that the μ opioid receptor can stimulate PLC activation andthat this coupling is blocked by M119.

To determine if other opioid receptors couple to PLC activation, opioidreceptor-dependent IP generation was examined in hKOR-CHO and hDOR-CHOcells. Treatment with U50,488 (10 μM), with or without M119 (10 μM) ornor-BNI (10 μM), the κ-selective antagonist, in the hKOR-CHO cells hadno significant effect on IP production over control treated cells (FIG.15B). In the hDOR-CHO cells neither DPDPE (10 μM) (FIG. 15C) norDeltorphin II (10 μM) (FIG. 15D) significantly increased IP generationover control treated cells. Treatment with M119 (10 μM) and theδ-selective antagonist, naltrindole (100 μM) also had no significanteffect (FIGS. 23C and 23D). Importantly, the levels of opioid receptorswere the same for all three cell lines (B_(max) values not significantlydifferent, data not shown). These in vitro data correlate with theeffects of M119 in vivo where only in the mice treated with thep-preferring compounds was a significant potentiation inantinocicieption observed. Thus, the selective effects of M119 observedin vivo on μ opioid receptor-dependent antinociception can be the resultof selective coupling of μ opioid receptor to Gβγ-dependent PLCactivation.

(3) M119 Attenuated Acute Morphine Antinociceptive Tolerance.

As an initial in vivo test of the efficacy of M119 in reducing thedevelopment of morphine antinociceptive tolerance, an acute toleranceassay was performed. This assay was used to measure tolerance because itrequires considerably less compound than traditional toleranceparadigms, while still yielding reliable results. The antinociceptiveeffect of repeated (0, 2 hr, 4 hr, 6 hr) doses (1-10 nmol) of morphineand the development of acute antinociceptive tolerance are shown herein.Morphine produced dose-dependent antinociception in the 55° C.warm-water tail-flick assay. The ED₅₀ value (95% CL) for i.c.v. morphineat time 0 was 0.59 nmol (0.16-2.2 nmol). After repeated administration,acute morphine antinociceptive tolerance developed by 4 hr with asignificant shift in the ED₅₀ value (p<0.01). At 4 hr, the ED₅₀ valuehad shifted 8-fold, 4.9 nmol (1.8-13 nmol) and by 6 hr, the ED₅₀ valuehad shifted 16-fold, 9.6 nmol (3.2-29 nmol) (p<0.001). Co-administrationof M119 with morphine greatly attenuated acute morphine antinociceptivetolerance. The ED₅₀ values remained virtually unchanged, with nostatistical difference, from time 0, 0.16 nmol (0.02-1.3 nmol) to the 6hr time-point, 0.22 nmol (0.008-5.7 nmol). Control mice were treatedunder the same injection protocol, however, received only vehicle. Nosignificant change from baseline tail-withdrawal values were observedfor any of the control mice at any of the times tested, indicating thatthe procedure employed in the acute tolerance protocol was notcontributing to the data which was observed.

(4) Pre-treatment With M119 Attenuated Acute Morphine PhysicalDependence.

In an acute model of morphine dependence, mice were treated withmorphine (100 mg/kg, s.c., −4 hr), vehicle (i.c.v.) 30 min prior tonaloxone, then administered naloxone (10 mg/kg, i.p.) which resulted inwithdrawal jumping. These mice jumped an average of 71±12 times in the15-min counting period. To test if M119 would attenuate acute morphinewithdrawal, mice were treated with morphine (100 mg/kg, s.c., −4 hr) andadministered M119 (100 nmol, i.c.v.) 30 min prior to the administrationof naloxone (10 mg/kg, i.p.). Under this test condition, mice jumpedsignificantly less times in the 15-min counting period, an average of23±14 compared to 71±12 for the control mice. This indicates that M119blocks the development of physical symptoms associated with opiatedependence. Post hoc analysis (Student's t-test) indicated thatco-administration of M119 with naloxone produced significantly lessvertical jumps than naloxone alone (p<0.05).

c) Discussion

Herein, the novel approach of using a Gβγ-inhibitor to influence theefficacy and potency of morphine in several animal models of μ-opioidreceptor function is disclosed. The mechanism of action of this compoundis based on studies demonstrating that the Gβγ-dependent enzyme, PLCβ3,was a negative modulator of μ opioid-receptor signaling both in PLCβ3knock-out mice and in dorsal root ganglion neurons. It was demonstratedherein that the Gβγ inhibitor, M119, inhibited PLCβ3 in vitro and thatconcomitant administration of this novel drug with morphine in vivoresulted in a 10-fold shift in morphine analgesic potency, virtuallyidentical, to data from the PLCβ3 knock-out studies (Xie et al., 1999,Bonacci et al., 2006). Herein, the specificity and underlying mechanismsof action of M119 in potentiating analgesia are further explored.Additionally, evidence was presented extending the therapeutic efficacyof this approach.

M119 Displayed μ Receptor-Selective Specificity In Vivo. Increasedanalgesic potency was observed in mice treated with M119 and morphineand also M119 and the receptor-selective agonist, DAMGO. Minimal effectswere observed with κ- and δ-selective agonists, U50,488, DPDPE, andDeltorphin II. These studies indicated that M119 was selective, in vivo,for the potentiation of μ-mediated analgesia. Similar results wereobtained from the in vitro IP assay, with only the μ-agonists, DAMGO andmorphine, stimulating IP generation which was inhibited by M119. The κand δ opioid receptors do not activate the same PLC isoforms, or are notacting in the same brain regions, and therefore are not inhibited by thesame PLC-dependent feedback pathway observed for the μ-opioid receptorand it is for this reason that that M119 lacks efficacy with thesereceptors.

Galeotti and colleagues (2006) recently demonstrated the importance ofPLCβ3 in opposing morphine analgesia utilizing antisense phosphodiesteroligonucleotides specifically to PLCβ3. In mice treated both withmorphine and the antisense oligonucleotide, a potentiation in analgesicresponse was observed (Galeotti et al., 2006). The same group alsodemonstrated localization of PLCβ3 in regions of the brain important fornociceptive transmission which have been previously shown to alsoexpress the μ opioid receptor (Galeotti et al., 2006).

(1) Downstream Signaling And The Role Of Phospholipids In Tolerance andDependence.

There is evidence that the PLC pathway can influence the development ofopioid tolerance and dependence. Inhibitors of PLC (Smith et al., 1999),IP₃ receptors (Smith et al., 1999), and PKC (Bilsky et al., 1996; Smithet al., 1999; Bohn et al., 2002) all attenuated morphine tolerance. PKCknock-out animals also exhibited attenuated morphine tolerance (Zeitz etal., 2001). It has also been suggested that generation of IP₃ and DAGalong with activation of PKC may be important for the development ofopioid-dependence (Fundytus and Coderre, 1996; Smith et al., 1999).Herein, it was shown that M119 attenuated both acute antinociceptivetolerance and dependence but which of these specific mechanismsdownstream of βγ-dependent PLCβ3 regulation are responsible for theseeffects remains to be defined.

4. Example 4 Small Molecule Targeting of G-Protein Beta Gamma inCardivoscular Disease

Bioavailable Gβγ-GRK2 inhibitory compounds with exciting data have beenidentified. Therefore, selective small molecule compound targeting ofGβγ-GRK2 is a novel therapeutic paradigm for HF, and selective Gβγcompounds elucidates important β-AR-Gβγ pathways in HF.

Heart failure (HF) continues to be the leading cause of death worldwide,having surpassed infectious disease in the 1990s: it has recently beenpredicted that HF will also become the leading cause of all disabilityby 2020(Murray C J, Lopez A D. Lancet 1997; 349(9064):1498-1504).Current data indicate that 5-year survival following the diagnosis of HFis 50%, and that 1-year survival for those with end-stage disease isonly 50% (Califf R M, et al. Am Heart J. 1997; 134:44-54), despitesubstantial therapeutic advances in the past two decades. HF is theleading cause for hospitalization in the U.S. at the cost of $300billion per year. For patients with end-stage HF, there are few optionsfor effective treatment. Although cardiac transplantation is the mosteffective treatment for end-stage HF, substantial limitations of thissurgical intervention include an extremely limited supply of acceptabledonor hearts, reaching a plateau of ˜2000/year in the USA, <3000/yearworldwide.

(1) 62 -Adrenergic Receptor Signaling in the Heart

G-protein coupled receptors (GPCRs) play an important role in both localand systemic regulation of heart function. In particular, β-adrenergicreceptors (β-AR) play a critical role in regulating cardiaccontractility, including both chronotropy and inotropy. HF is associatedwith chronic down-regulation and desensitization of cardiac β-ARs, duein part to chronic agonist stimulation(Bristow M R, et al. N Engl J Med.1982; 307:205-211; Rodman H A, et al. Nature. 2002; 415(6868):206-212).Attenuation and desensitization of β-AR signaling and responsiveness ismediated by the β-AR kinase ((βARK1). βARK1 is a member of the Gprotein-coupled receptor (GPCR) kinase (GRK) family, and is also knownas GRK2. GRK2 is a cytosolic enzyme that targets and phosphorylatesagonist-occupied GPCRs, including myocardial β-ARs, via recruitment byand binding to the βγ-subunits of heterotrimeric G-proteins (Gβγ)following GPCR agonist stimulation (Koch W J, et al. Annu Rev Physiol.2000; 62:237-26). Agonist-stimulated Gβγ-GRK2 interaction is aprerequisite for GRK2-mediated GPCR (including β-AR) phosphorylation,homologous receptor desensitization and subsequent internalization anddegradation (Bristow M R, et al. N Engl J Med. 1982; 307:205-211).

Elevated expression and activity of the G-protein coupled receptorkinase (GRK2, a.k.a. β-AR kinase, βARK1) is a hallmark of human andexperimental animal HF (Rockman H A, et al. Nature. 2002;415(6868):206-212; Hansen J L, et al. Trends Cardiovasc Med. 2006;16(5):169-177). Furthermore, cardiac targeted overexpression of GRK2 candirectly cause HF in experimental animal models (Koch W, et al. Science.1995; 268:1350-1353). Importantly, levels of GRK2 expression andactivity from cardiac tissue and circulating lymphocytes correlatedirectly with the severity of human HF: it was found GRK2 expression andactivity were elevated in end-stage HF, and were normalized followingsalutary left ventricular assist device (LVAD) support (FIG. 16 and(Blaxall B C, et al. J Am Coll Cardiol. 2003; 41(7):1096-1106; Hata J A,et al. J Card Fail. 2006; 12(5):360-368).

(2) A Role for G βγ Subunit Inhibition in the Heart

Since Gβγ binding is a critical prerequisite for GRK2-mediated GPCRdesensitization, several approaches have been explored to interdict theGβγ-GRK2 interaction. GRK2 possesses three general domains, including anN-terminal RGS and protein recognition domain, a central kinase domain,and a C-terminal region encoding the Gβγ binding domain. To study therole of the Gβγ binding domain in the functional regulation of GRK2, theC-terminal 197 amino acids encoding the GRK2 Gβγ binding domain (βARKct)was expressed in cells as a Gβγ peptide inhibitor of GRK2, where itattenuated homologous γ-AR desensitization in a GPCR-specific manner(Koch W J, et al. J Biol Chem. 25 1994; 269(8):6193-6197). Subsequently,transgenic mice were created with myocardial targeted expression ofβARKct, which demonstrated enhanced basal cardiac function and responseto isoproterenol (Koch W J, et al. Science. 1995; 268(5215):1350-1353).Mating of the cardiac-targeted βARKct mice with the GRK2 overexpressingmice normalized cardiac function, providing direct evidence that themechanism responsible for the phenotype in these mice was βARKctinhibition of G_(βγ)-mediated signaling, including GRK2 activity(Koch W,et al. Science. 1995; 268:1350-1353; Akhter S A, et al. Circulation.1999; 100(6):648-653).

To determine the direct role of GRK2 and the Gβγ-GRK2 interaction in thepathogenesis of HF, the cardioprotective potential of βARKct has beenassessed in animal models of HF. The data repeatedly demonstrate asalutary, cardioprotective effect of βARKct both by transgenesis ingenetic models of HF, as well as through adenoviral delivery in surgicalmodels of HF (Blaxall B C, et al. Physiol Genomics. 2003; 15(2):105-114;Rockman H A, et al. Proc Natl Acad Sci USA. 1998; 95(12):7000-7005; KochW J. Mol Cell Biochem. 2004; 263(1-2):5-9). Notably, βARKct has not onlynormalized cardiac function, but has also normalized aspects of β-ARsignaling(Blaxall B C, et al. Physiol Genomics. 2003; 15(2):105-114;Rockman H A, et al. Proc Natl Acad Sci USA. 1998; 95(12):7000-7005;Harding V B, et al. Proc Natl Acad Sci USA. 2001; 98(10):5809-5814).Furthermore, βARKct was shown to be synergistic with β-AR blockers(standard medical therapy for HF) in the cardiac calsequestrinoverexpressor (CSQ) mouse model of HF. Importantly, βARKct has also beenshown to normalize contractile function of failing human cardiacmycoytes (Williams M L, et a;. Circulation. 2004; 109(13):1590-1593).Thus, inhibition of the Gβγ-GRK2 interaction possesses great therapeuticpromise.

The therapeutic possibilities of targeting Gβγ signaling in thepathogenesis of HF were further validated in a recent report by theUngerer group(Li Z, et al. Gene Ther. 2003; 10(16):1354-1361). Thisgroup investigated an alternative dominant-negative peptide approach tointerdicting Gβγ signaling in direct comparison to βARKct. Phosducin isa Gβγ binding protein first discovered in retinal tissue followingactivation of the GPCR transducin, with phosducin homologues nowdescribed in several tissues. Like βARKct, phosducin also translocatesfrom cytosol to membrane upon GPCR activation, binds Gβγ subunits withhigh affinity, and inhibits subsequent Gβγ signaling events, includingGRK2 recruitment (Bauer P H, et al. Nature. 1992; 358(6381):73-76;Gaudet R, et al. Cell. 1996; 87(3):577-588; Hekman M, et al. FEBS Lett.1994; 343(2):120-124; Schulz R. Pharmacol Res. 2001; 43(1):1-10). Priorreports had indicated more stable high affinity of an ˜200 amino acidN-terminally truncated version of phosducin (nt-del-phd). Viral genedelivery of either βARKct or nt-del-phd delivered after pacing-inducedHF in rabbits equally normalized cardiac function in vivo. Bothtransgenes also normalized contractility of isolated failingcardiomyocytes, with some differences in their effects on β-ARsignaling. Taken together, the above data strongly indicate thetherapeutic potential of targeting Gβγ signaling in HF.

(3) β-AR-Gβγ-PI3Kγ Signaling in the Heart

Cardiac Gβγ signaling results in activation of downstream pathwaysbeyond GRK2, including phosphoinositide 3-kinase g (PI3Kγ), the onlyPI3K regulated in part by Gβγ signaling⁵. Following β-AR stimulation,cytosolic GRK2 interacts with phosphoinositide 3-kinase γ (PI3Kγ).Gβγ-mediated membrane recruitment of GRK2 appears to recruit PI3Kγ tothe membrane proximal to β-AR, where both the protein and lipid kinaseactivity of PI3K are required for the pathologic β-AR receptordesensitization and downregulation observed in HF (Naga Prasad S V, NatCell Biol. 2005; 7(8):785-796; Naga Prasad S V, et al. J Cell Biol.2002; 158(3):563-575; Naga Prasad S V, et al. J Biol Chem. 2001;276(22):18953-18959; Perrino C, et al. Vascul Pharmacol. 2006;45(2):77-85).

A growing body of evidence demonstrates that displacement of PI3Kγ fromthe GRK2 complex is cardioprotective, in part through reducing β-ARdesensitization and down-regulation. Experiments have includedcardiac-targeted expression of a kinase-inactive PI3Kγ(PI3Kγ_(inact))(Nienaber J J, et al. J Clin Invest. 2003;112(7):1067-1079; Perrino C, et al. J Am Coll Cardiol. 2005;45(11):1862-1870; Perrino C, et al. J Clin Invest. 2006;116(6):1547-1560) or a large peptide inhibitor of the protein kinasedomain of PI3K(Perrino C, et al. J Am Coll Cardiol. 2005;45(11):1862-1870; Curcio A, et al. Am J Physiol Heart Circ Physiol.2006; 291(4):H1754-1760; Perrino C, et al. Circulation. 2005;111(20):2579-2587). Both large peptides disrupt the cytosolic GRK2-PI3Kγinteraction and normalize β-AR signaling and cardiac function both invivo and in isolated cardiomyocytes from chronic HF models. Despiteearly suggestions of mild cardioprotection in PI3Kγ null animals(Crackower M A, et al. Cell. 2002; 110(6):737-749; Oudit G Y, et al.Circulation. 2003; 108(17):2147-2152), subsequent experiments haveconvincingly demonstrated their increased susceptibility to β-ARabnormalities and cardiac injury in multiple HF models. Concurrentexperiments showed that PI3Kγ_(inact) and PIK domain animals werecardioprotective and normalized β-AR signaling in these HF models. Theseexperiments suggest that β-AR-Gβγ-GRK2-PI3Kγ interactions in the heartlead to cardiac pathophysiology, yet there are important aspects ofPI3Kγ signaling in the heart that should be maintained. In summary, theβ-AR-Gβγ-GRK2-PI3Kγ cascade plays an important role in cardiacpathophysiology and pathologic β-AR abnormalities. What remains unclearis whether PI3Kγ-mediated regulation of β-AR signaling is Gβγ- and/orGRK2 dependent or independent.

Targeting Gβγ signaling has proven a promising therapeutic paradigm inthe treatment of HF. Unfortunately, despite vast efforts in bothacademia and industry, therapeutic targeting of cardiac Gβγ in HF todate has only been achieved by ˜200 amino acid peptides that can only beadministered via transgenesis or viral gene therapy, which facessubstantial hurdles in development as a therapeutic modality.Identification of selective and differential small molecule compoundstargeting Gβγ signaling in the pathogenesis of HF provides valuable newresearch tools to dissect the β-AR-Gβγ-GRK2-PI3Kγ signaling pathway(s),and may provide novel and readily bioavailable therapeutics forcardiovascular disease.

b) Results

The cardioprotective peptide inhibitors described above (βARKct,nt-del-phd, PI3Kγ_(inact)) function in part by inhibiting aspects of Gβγsignaling, including recruitment of GRK2 (and PI3K) to Gβγ followingβ-AR agonist stimulation (a requisite step in GRK2-mediateddesensitization of agonist occupied β-ARs). These data highlight thetherapeutic potential of Gβγ inhibitors in HF, but are hampered by thefact that all are peptides requiring viral vector delivery fortherapeutic efficacy. Thus, identification of small molecule compoundsthat inhibit Gβγ is a promising and effective therapeutic approach forHF.

(1) Chemical Gβγ Inhibitor Studies in Isolated Adult MouseCardiomyocytes

Data obtained with purified proteins indicated that one of the highaffinity Gβγ binding compounds, M119, could inhibit the Gβγ-GRK2interaction (Bonacci T M, et al. Science. 2006; 312(5772):443-446).Subsequently, it was found that M119 demonstrated the capability toreduce GRK2 membrane recruitment following fMLP stimulation of leukocytecells. Thus, it was tested whether M119 could block GRK2 recruitment tomembrane-bound Gβγ subunits upon agonist stimulation of β-AR incardiomyocytes, similar to what had been observed in vitro with thepeptide inhibitors described above. Indeed M119 reduced β-AR-mediatedrecruitment of GRK2 to membranes of isolated adult mouse cardiomyocytes,with a mild reduction of membrane associated GRK2 at baseline.

To assess whether the M119 inhibition of Gβγ-mediated GRK2 recruitmentresulted in downstream effects on β-AR signaling, β-AR mediatedactivation of adenylyl cyclase and subsequent cAMP generation wasdetermined in isolated adult mouse cardiomyocytes. Isolatedcardiomyocytes were treated with vehicle (V) or 100 nM isoproterenol (I)for 15 minutes in the presence or absence of a 5 minute pre-treatmentwith 10 μM M119 (M). Forskolin treatment, which directly activatesadenylyl cyclase, was used as a control. It was found that M119concomitantly reduced GRK2 recruitment to the membrane and enhanced cAMPgeneration both at baseline and in response to the β-AR agonistisoproterenol. These data indicated that M119 was blocking Gβγ-mediatedGRK2 recruitment, and was effecting changes similar to those found withthe salutary Gβγ inhibitory peptides.

The above data led to investigations that M119 plays a positivefunctional role in the heart, due in part to its inhibition of Gβγ-GRK2interactions. Prior to pursuing in vivo studies with M119, the role ofM119 affecting contractility was evaluated by assessment of isolatedadult mouse cardiomyocyte contractility at baseline and in response toβ-AR stimulation. In agreement with the signaling data, it was foundthat M119 enhanced cardiomyocyte contractility both at baseline and inresponse to β-AR treatment with the β-AR agonist isoproterenol.

In addition to cardiomyocyte contractility studies with M119,contractility studies were also conducted with the inactive form of M119(M119b). M119b demonstrated no effect on cardiomyocyte contractility atbaseline or in response to contraction elicited by isoproterenoltreatment. Furthermore, a compound closely related to M119, referred toas both DL-382 and its trade name Gallein, was investigated in thesecontractility studies. These molecules bear a similar chemical scaffoldwith minor differences in the fourth ring structure. The studiesdemonstrated that closely related compounds to M119, such as Gallein,produce significant positive effects on isolated cardiomyocytecontractility at concentrations similar to M119.

(2) Chemical Gβγ inhibitor Studies in an Acute Mouse Model of HF

Based on the positive cardiomyocyte data, the cardiac effects of M119were tested in vivo. The NCI compound database and chemical library wasoriginally assembled to investigate these compounds utilizing both ayeast high-throughput screening assay as well as in vivo compounddelivery to mice for 1-2 weeks. The NCI had delivered M119 at a dose of250 mg/kg/day to animals for two weeks with no reported overt evidenceof toxicity. Based on this data, the effects of M119 were investigatedon an acute model of HF representative of the chronic catecholaminestress and adrenergic dysfunction found in human HF. Wild type mice wereimplanted with mini-osmotic pumps filled with either vehicle or thenon-selective β-AR agonist isoproterenol. These pumps release a constantfluid volume, and 30 mg/kg/day of isoproterenol (Iso) has been used toattain pathologic cardiac hypertrophy and dysfunction in one weekImmediately following mini-osmotic pump implantation of vehicle or Iso(30 mg/kg/day), mice from each group were injected intraperitoneal (IP)once daily with equal volumes of either vehicle or a moderate dose ofM119 (100 mg/kg) for seven days. Conscious echocardiography andpost-mortem morphology demonstrated that M119 alone had no effect oncardiac function or morphology. The Iso pumped animals indeed suffered adecrease of cardiac function coupled with pathologic hypertrophy.Excitingly, the Iso pumped animals treated for one week with dailyintraperitoneal injections of M119 demonstrated near normalization ofcardiac function and morphology (FIG. 17), and a reduction ininterstitial cardiac fibrosis. Finally, it was found that M119 treatmentnearly normalized GRK2 expression in the Iso HF model (FIG. 18).Together, these data further validate an important role for Gβγsignaling in the heart, and indicate the therapeutic potential of Gβγinhibitory compounds in the treatment of HF.

(3) Other Compounds

Compound M119 appears to block Gβγ-activated GRK2 and PI3K binding andactivity in vitro. In contrast, compound M201 appears to block Gβγ-GRK2interactions, but potentiates PI3K activity in vitro (Bonacci T M, etal. Science. 2006; 312(5772):443-446). Importantly, compounds were foundthat differentially block Gβγ interactions with GRK2 and/or PI3K invitro. These compounds offer substantial delivery, size (˜400 Da, vs.˜35 kDa peptides) and specificity advantages over the large peptides,and provide powerful tools to dissect the β-AR-Gβγ-GRK2-PI3K signalingpathway and define the role of specific components in pathologic β-ARdesensitization and HF.

In summary, the data demonstrate that Gβγ-GRK2 inhibitory compounds area very promising therapeutic paradigm for the treatment of HF. Inparticular, the bioavailable and cell permeable M119 Gβγbinding/blocking compound can: 1) reduce GRK2 membrane recruitment uponβ-AR stimulation in isolated adult cardiomyocytes; 2) enhance basal andβ-AR mediated cAMP generation in isolated adult cardiomyocytes; 3)enhance both basal and β-AR stimulated isolated cardiomyocytecontractility; 4) reduce pathologic hypertrophy in a chronic β-ARagonist model of HF; and 5) normalize cardiac function as measured byechocardiography in this model of HF. Herein, newly identifiedGβγ-signal specific chemical inhibitors are tested to determine whetherGβγ-sensitive PI3K regulation of β-AR signaling is GRK2 dependent orindependent (FIG. 19).

(4) Toxicity

Toxicity studies of M119 were undertaken where a one week dosing regimenof 100 mg/kg/day of M119 delivered by intraperitoneal injection. Here,no overt toxicity was observed nor any overt toxicity in intact animalsor upon histological examination of lung, liver, brain, heart and kidneyfollowing two weeks of daily Gallein injections at doses of 10, 30 and100 mg/kg/day.

(5) Alpha2-Adrenergic Receptors and Catecholamine Release

A beneficial effect of inhibiting G-protein-beta-gamma mediateddesensitization of alpha2-adrenergic receptors in the adrenal gland hasbeen shown. Inhibiting this desensitization restores a normal feedbackinhibition of catecholamine release that is disrupted in advanced heartfailure. Not only are the methods presented herein capable ofinvestigating adrenal chromaffin cell catecholamine release, but alsoM119 can reduce or prevent alpha2-adrenergic receptors in these cells,thus restoring normal levels of catecholamine release from the adrenalgland. Thus, dual inhibition of cardiac and adrenal adrenergic receptordesensitization with a systemically delivered agent is duallyefficacious in the treatment of heart failure.

c) Methods

(1) Determine in vivo Efficacy of M119 in Chronic Animal Models of HF,Including Effects on β-AR Signaling

HF is associated with chronic β-AR desensitization and down-regulation,due in part to Gβγ-mediated recruitment of GRK2, which demonstratesenhanced expression and activity in HF. Compounds, including M119, havebeen identified which inhibit the Gβγ-GRK2 interaction in vitro. Usingadult mouse cardiomyocytes, it was found that M119 reduces β-ARstimulated membrane recruitment of GRK2, and enhances cAMP generation atbaseline and in response to β-AR stimulation. Also demonstrated wasincreased cardiomyocyte contractility at baseline and in response toβ-AR agonist. Importantly, normalization of cardiac function, morphologyand GRK2 expression was shown in an acute animal model of HF.Previously, large peptide inhibition of the Gβγ-GRK2 interaction withβARKct has enhanced baseline and Iso-stimulated in vivo hemodynamics.Further, βARKct has normalized cardiac function and β-AR signaling inboth the cardiac calsequestrin (CSQ) overexpressor HF mouse as well asfollowing transverse aortic constriction, and has demonstrated similarmortality benefit to and synergy with β-blocker therapy, currentstandard treatment for HF patients (Harding V B, et al. Proc Natl AcadSci USA. 2001; 98(10):5809-5814; Tachibana H, et al. Circulation. 2005;111(5):591-597). Importantly, CSQ animals replicate hallmark β-ARabnormalities seen in human HF. Disclosed herein, M119 can normalizeβ-AR-Gβγ signaling and cardiac function in vivo and in a long-term HFmodel, both alone and in combination with β-blocker therapy.

(a) Hemodynamic Characterization of M119 in Basal wt Animals

To determine the hemodynamic response of wt mice in response to M119,invasive in vivo hemodynamics are determined both at baseline and inresponse to increasing doses of the β-AR agonist Iso, in the absence andpresence of a 100 mg/kg IP dose of M119 delivered four hours prior tohemodynamic assessment, or with bolus M119 (10 mg/kg) deliveredconcurrent with the hemodynamic studies at baseline and in response toincreasing doses of Iso. Also disclosed herein M119 enhancesbasal andIso-induced cardiac contractility.

(b) Investigation of M119 in a Chronic HF Model

Cardiac-specific calsequestrin (CSQ) mice provide a close representationof the development and progression of human HF, including progressivedilated cardiomyopathy and associated β-AR dysfunction.

The CSQ mice develop dramatic dilated cardiomyopathy by 10-14 weeks. Theanimals are treated with the β1-AR selective β-blocker metoprolol 2mg/mL in drinking water (˜350 mg/kg/day) for breeding purposes, and havefound no adverse effects. These are the same concentrations utilized inthe experiments that compared metoprololol to βARKct in the CSQ mice¹⁶.To test the in vivo effects of M119 alone or in combination withβ-blocker therapy, four groups of male CSQ mice are tested beginning atone month of age: 1) regular drinking water; daily vehicle injection IP,2) 2 mg/mL metoprololol in drinking water, daily IP vehicle injection,3) regular drinking water, daily M119 injection (100 mg/kg/day) IP, and4) 2 mg/mL metoprololol in drinking water, daily M119 injection (100mg/kg/day) IP. Additionally, M119 demonstrates greater effects thanmetoprolol on β-AR signaling, cardiac dysfunction and survival, and thatM119 is synergistic with metoprolol in improving these parameters.

(c) Serial Echocardiography.

Conscious echocardiography is non-invasive, can be performed in serialfashion, and provides excellent data regarding in vivo cardiacmorphology and function. Furthermore, the echocardiography data indicateM119 is cardioprotective in an acute model of HF. Mice are analyzed atbaseline at one month of age and weekly for the duration of thepharmacological treatments described above.

(d) Heart Weight and LV to Body Weight Ratio.

To determine the extent of hypertrophy/dilation heart weight, and LV(mg) divided by body weight (g) ratio are examined upon sacrifice ofanimals after 1 month of treatment, and normalized with M119 in allconditions.

(e) Analysis of Fetal Gene Expression.

Heart failure is associated with the expression of a variety of thegenes present in the heart during embryonic development. These genesinclude ANF, BNP, α-skeletal actin, and β-MHC. The expression of thesegenes are analyzed in mice using real-time PCR analysis (5 mice pergroup).

(f) β-AR Binding and Adenylyl Cyclase Assays.

To determine the β-AR effect of M119 in HF, alone or in combination withmetoprolol, the total and sub-type specific membrane β-AR expression andadenylyl cyclase activity is assessed in LV homogenates (SpecificMethods), and anticipate normalization of both with M119.

(g) GRK2 Expression and Activity.

Elevated expression and activity of GRK2 is a hallmark of HF, andconcordance between these parameters and the severity of HF has beendemonstrated, including response to therapy (Hata J A, et al. J CardFail. 2006; 12(5):360-368). Total GRK2 expression and activity isdetermined in LV homogenates, and anticipate normalization with M119.

(h) Histochemistry.

HF in the CSQ model is associated with structural myocardialabnormalities and increased fibrosis. To assess the role of M119 inthese parameters, and to corroborate parameters found byechocardiography, hematoxylin and eosin staining (H&E), is performed andwall thickness determined, as well as, myocyte structure andcross-sectional area. To assess fibrosis, the Masson's Trichrome andtotal collagen stain are used.

(i) Apoptosis.

Apoptotic cell death contributes to the heart remodeling and developmentof dilated cardiomyopathy, particularly after myocardial infarction. Theimpact of M119 expression on cardiac apoptosis is determined using twodifferent methods: Tunel staining on heart sections, and determinecaspase-3 activity in LV homogenates with a colorimetric assay, as wellas Bax expression and Caspase 3 cleavage by Western Blot.

(2) Determine the Role of Targeted, Specific Small Molecule Gβγ Blockadeof GRK2 or PI3Kγ Alone or in Combination in β-AR Signaling andCardiomyocyte Contractility.

Several lines of evidence indicate that β-AR-Gβγ-GRK2-PI3Kγ interactionsand signaling in the heart lead to β-AR dysfunction and cardiacpathophysiology, and that there are important aspects of PI3Kγ signalingin the heart that should be maintained. The data, derived almostexclusively from large peptide interference, has indicated an importantrole for Gβγ-mediated GRK2 recruitment to the membrane, and for PI3Kγrecruitment to GRK2, prior to their role in pathologic β-ARdesensitization. What remains unclear is the specificity of these largepeptide inhibitors, whether blockade of Gβγ binding to GRK2 or PI3Kγalone can prevent β-AR dysfunction and HF, and whether PI3Kγ-mediatedregulation of β-AR signaling is Gβγ- and/or GRK2 dependent orindependent. Compounds were identified in vitro that offer substantialdelivery, size (˜400 Da, vs. ˜35 kDa peptides) and potential specificityadvantages over large peptide inhibitors. These compounds, at similarEC₅₀ concentrations of 1-12 μM: 1) block Gβγ-binding and activation ofGRK2 and PI3K (M119), 2) block Gβγ-GRK2 binding and activity, but do notblock Gβγ-PI3K, and actually potentiate PI3K activity (M201 (Bonacci TM, et al. Science. 2006; 312(5772):443-446)), 3) block only Gβγ-GRK2binding and interaction with no effect on PI3Kγ binding or activity, 4)block only Gβγ-mediate PI3Kγ activity with no effect on GRK2, and 5)block neither GRK2 nor PI3Kγ binding nor activity associated with Gβγ(M119b (Bonacci T M, et al. Science. 2006; 312(5772):443-446).

Experiments in this aim test the hypothesis that targeted Gβγ-GRK2inhibition is key to the normalizing β-AR signaling and cardiaccontractility. The Gβγ-signal specific chemical inhibitors are used totest whether Gβγ-sensitive PI3K regulation of β-AR signaling is GRK2dependent or independent. These experiments provide new tools to dissectβ-AR-Gβγ-GRK2-PI3K signaling.

(3) Determination of Gβγ-GRK2 and Gβγ-PI3Kγ Signal-Specific Effects onChronic β-AR Signaling

To assess cardiomyocyte-specific effects of the specific compounds β-ARsignal transduction, the signal specific effects of each of thecompounds are examined in neonatal rat ventricular myocytes (NRVM),where it was demonstrated pathologic β-AR dysfunction following chronicβ-AR agonist treatment with Iso(Ding B, et al. Circulation. 2005;111(19):2469-2476; Ding B, et al. Proc Natl Acad Sci USA. 2005;102Milano C A, et al. Proc Natl Acad Sci USA. 1994;91(21):10109-10113:14771-14776). NRVMs are isolated, cultured, thentreated with 10 μM Iso for 10 minutes (localization studies) or 24 hoursto produce pathologic β-AR desensitization and down-regulation, in thepresence and absence of the following compounds at concentrations of 1and 10 μM: 1) M119, (blocks Gβγ-binding and activation of GRK2 andPI3K), 2) M201 (blocks Gβγ-GRK2 binding and activity, Gβγ-PI3K:potentiates PI3K activity), 3) Mcmpd (blocks only Gβγ-GRK2 binding andinteraction with no effect on PI3Kγ binding or activity), 4) Mcmpd(blocks only Gβγ-mediated PI3Kγ activity with no effect on GRK2), and 5)M119b (blocks neither GRK2 nor PI3Kγ binding nor activity associatedwith Gβγ). In parallel, NRVMs are adenovirally infected with GRK2,βARKct, PIK, or PI3Kγ_(inact) (kindly provided by Drs. Walter Koch andHoward Rodman, see letters) and treated in the presence and absence of10 μM Iso for comparison of effect and specificity. Following 24 hoursof treatment, cells undergo characterization described below.

(a) β-AR Binding.

Total and sub-type specific membrane β-AR is determined in membraneisolates of NRVMS following 24 hrs treatment described above (SpecificMethods).

(b) Whole Cell Adenylyl Cyclase.

Cells are harvested after 24 hrs, and whole cell adenylyl cyclase assaysdetermine β-AR-, NaF- and forskolin-stimulated cAMP generation (SpecificMethods).

(c) GRK2 Expression, Localization and Activity.

Whole cell lysates are used to assess effects on total GRK2 expressionafter 24 hours of treatment by Western Blotting. Membrane and cytosolicfractionation are performed to assess GRK2 membrane localizationfollowing either 10 minutes or 24 hours of treatment by WesternBlotting. GRK2 activity is determined after 24 hrs of treatment(Specific Methods).

(d) Membrane- and GRK2-Associated PI3K Activity.

Following 24 hrs treatment, membrane fractions are utilized to determinetotal membrane-associated PI3K activity. Membrane fractions areimmunoprecipitated with anti-GRK2 antibody to assess GRK2-associatedPI3K activity (Specific Methods).

(4) Determination of Gβγ-GRK2 and Gβγ-PI3Kγ Signal-Specific EffectsCardiomyocyte Contractility

(a) Adult Mouse Cardiomyocyte Contractility.

To assess effects of the signal-specific Gβγ-inhibitory compoundsdescribed above, the established method of contractility assessment isused in isolated adult mouse cardiomyocytes. Following isolation andplating, contractile assessment of cardiomyocytes is performed in theabsence and presence of the β-AR agonist Iso (100 nM), in the absenceand presence of the individual Gβγ inhibitory compounds described aboveat 1 and 10 μM (Specific Methods).

(b) Morphological and Histological Characterization of Transgenic MouseHearts:

Morphological and histological examination as previously described(Rockman H A, et al. Proc Natl Acad Sci USA. 1998; 95(12):7000-7005;Milano C A, et al. Proc Natl Acad Sci USA. 1994; 91(21):10109-10113;Jaber M, et al. Proc Natl Acad Sci USA. 1996; 93(23):12974-12979;Maekawa N, et al. Circulation. May 22 2006; Maekawa N, et al. J Am CollCardiol. 2002; 39(7):1229-1235; Itoh S, et al. Circulation. 2006;113(14):1787-1798), including hematoxylin and eosin (H&E) Masson'strichrome and total collagen staining following standard techniques(Rockman H A, et al. Proc Natl Acad Sci USA. 1998; 95(12):7000-7005;Kypson A P, et al. J Thorac Cardiovasc Surg. 1998; 115(3):623-630).

(c) Determination of β-AR Density:

Myocardial membranes are prepared, assayed with [¹²⁵I]-CYP, non-specificbinding determined with 1 μM alprenolol. Specific binding (B_(max)) isnormalized to membrane protein concentration (Koch W J, et al. Science.1995; 268(5215):1350-1353; Milano C A, et al. Proc Natl Acad Sci USA.1994; 91(21):10109-10113). Sub-type specific β-AR density is determinedby competition binding, % of high affinity and low-affinity receptors isdetermined (Koch W J, et al. Science. 1995; 268(5215):1350-1353).

(d) Adenylyl Cyclase Assays:

Myocardial membranes are prepared as described above. Adenylyl cyclaseactivity under basal conditions and in response to 100 μM isoproterenol,10 mM NaF or 100 mM forskolin is determined as described(Koch W J, etal. Science. 1995; 268(5215):1350-1353; Milano C A, et al. Proc NatlAcad Sci USA. 1994; 91(21):10109-10113). Whole cell adenylyl cyclaseassays (cardiomyocyte) are performed with Assay Designs cAMP generationcolorimetric (EIA) kit (Ann Arbor, Mich.) per manufacturer instructions.

(e) In vivo Hemodynamic Measurements:

Performed as previously described (Wang H, et al. Circ Res. 2005;97(12):1305-1313). Adult mice are anesthetized and intubated.Hemodynamic measurements are recorded using a 1.0F high-fidelitymicromanometer Millar carotid catheter secured in the LV. Pressurerecordings are taken at baseline and 45 to 60 s after injection of 50 uLsaline or incremental doses of Iso via jugular cannulation.

(f) Conscious Echocardiography:

Performed as previously described. Briefly, the chest of conscious miceis shaved, the mice are restrained, acclimatized and transthoracicechochardiography performed with the VisualSonics Vevo 770 system(VisualSonics, Toronto, ON) designed specifically for rodent studies(NgC M, et al. J Clin Invest. 2004; 114(11):1586-1592; Zhou Y Q, et al.Physiol Genomics. 2004;18(2):232-244).

(g) GRK Activity Assay:

Extracts are prepared from tissue samples as described above. GRKactivity is assessed in membrane and cytosolic fractions (100 to 150 mgprotein) by light-dependent phosphorylation of rhodopsin-enriched rodouter segment membranes, phospho-rhodopsin is visualized byautoradiography (Iaccarino G, et al. Eur Heart J. 2005;26(17):1752-1758; Iaccarino G, et al. Circulation. 1998;98(17):1783-1789).

(h) PI3K Activity Assay:

Assays were performed as described previously (Nienaber J J, et al. JClin Invest. 2003;112(7):1067-1079). Membrane fractions are assayeddirectly or following GRK2 immunoprecipitation for GRK2-associated PI3K.In vitro lipid kinase assays are performed using PtdIns-4,5-P2, lipidsare extracted, spotted on TLC plates and resolved chromatographicallyand subjected to autoradiography.

(i) Neonatal Rat Ventricular Cardiomyocyte Isolation.

Performed as described previously (Ding B, et al. Circulation. 2005;111(19):2469-2476; Ding B, et al. Proc Natl Acad Sci USA. 2005; 102Milano C A, et al. Proc Natl Acad Sci USA. 1994;91(21):10109-10113:14771-14776). More than 90% of cells were NRVM(positive for α-actinin). Adenovirus-mediated transfection efficiency inall the cardiomyocytes is 90% to 95% at MO1 0.1-1.

(j) Adult Cardiomyocyte Isolation:

Adult cardiomyocytes are isolated from collagenase digested hearts asdescribed previously (O'Connell T D, et al. Methods Mol Biol. 2006;357:271-296). Briefly, the heart is excised, cannulated, perfused anddigested with collagenase II, myocytes are filtered, sedimented, calciumis re-introduced followed by plating on laminin coated dishes.

(k) Cardiomyocyte Contractility and Ca⁺⁺ Assessment:

Mechanical properties of ventricular myocytes are assessed by anIonOptix Myocam system (IonOptix Inc., Milton, Mass., U.S.A.). Myocytesare superfused (at 25° C.) with Tyrodes, cells are pre-loaded in culturemedium for 20 minutes with Fura-2, for concurrent calcium flux andmyocyte contractility following field stimulation at 0.5 Hz.

(5) Statistical Analysis:

For single biochemical/physiological observations, students t-test areapplied to compare animal treatments. Multiple responses of variousphysiological and biochemical and assays are analyzed using one-way orrepeated measures ANOVA. Post-hoc analysis (ie. Newman-Keuls) isperformed if significance is achieved, using P<0.05 for all tests.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains. Thereferences disclosed are also individually and specifically incorporatedby reference herein for the material contained in them that is discussedin the sentence in which the reference is relied upon.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

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What is claimed is:
 1. A method for treating a disease or condition involving at least one G protein βγ subunit activity in a patient having the disease or condition, the method comprising administering to the patient an effective amount of an agent that interacts with at least one amino acid residue of the protein interaction site of the G protein β subunit, whereby the at least one activity of the G protein is modulated and the disease or condition is treated in the patient, wherein the disease or condition is selected from the group consisting of an inflammatory condition, a cardiovascular disease or condition, and a vascular disease or condition, wherein the agent comprises a compound of:

wherein R^(5c) is a unit selected from the group consisting of phenyl, 2-carboxyphenyl, 3-carboxyphenyl, 4-carboxyphenyl, 2,3-dicarboxyphenyl, 2,4-dicarboxyphenyl, 2,5-dicarboxyphenyl, 2,6-dicarboxyphenyl, 3,4-dicarboxyphenyl, 3,5-dicarboxyphenyl, 2,3,4-tricarboxyphenyl, 2,3,5-tricarboxyphenyl, 2,3,6-tricarboxyphenyl, 2,4,6-tricarboxyphenyl, 2,3,4,5-tetracarboxyphenyl, 2,3,4,6-tetracarboxyphenyl, 2,3,4,5,6-pentacarboxyphenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 2,3-difluorophenyl, 2,4-difluorophenyl, 2,5-difluorophenyl, 2,6-difluorophenyl, 3,4-difluorophenyl, 3,5-difluorophenyl, 2,3,4-trifluorophenyl, 2,3,5-trifluorophenyl, 2,3,6-trifluorophenyl, 2,4,6-trifluorophenyl, 2,3,4,5-tetrafluorophenyl, 2,3,4,6-tetrafluorophenyl, 2,3,4,5,6-pentafluorophenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 2,3-dichlorophenyl, 2,4-dichlorophenyl, 2,5-dichlorophenyl, 2,6-dichlorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl, 2,3,4-trichlorophenyl, 2,3,5-trichlorophenyl, 2,3,6-trichlorophenyl, 2,4,6-trichlorophenyl, 2,3,4,5-tetrachlorophenyl, 2,3,4,6-tetrachlorophenyl, 2,3,4,5,6-pentachlorophenyl, 2-bromophenyl, 3-bromophenyl, 4-bromophenyl, 2,3-dibromophenyl, 2,4-dibromophenyl, 2,5-dibromophenyl, 2,6-dibromophenyl, 3,4-dibromophenyl, 3,5-dibromophenyl, 2,3,4-tribromophenyl, 2,3,5-tribromophenyl, 2,3,6-tribromophenyl, 2,4,6-tribromophenyl, 2,3,4,5-tetrabromophenyl, 2,3,4,6-tetrabromophenyl, 2,3,4,5,6-pentabromophenyl, 2-iodophenyl, 3-iodophenyl, 4-iodophenyl, 2,3-diiodophenyl, 2,4-diiodophenyl, 2,5-diiodophenyl, 2,6-diiodophenyl, 3,4-diiodophenyl, 3,5-diiodophenyl, 2,3,4-triiodo-phenyl, 2,3,5-triiodophenyl, 2,3,6-triiodophenyl, 2,4,6-triiodophenyl, 2,3,4,5-tetraiodo-phenyl, 2,3,4,6-tetraiodophenyl, 2,3,4,5,6-pentaiodophenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, 2,3-dihydroxyphenyl, 2,4-dihydroxyphenyl, 2,5-dihydroxyphenyl, 2,6-dihydroxyphenyl, 3,4-dihydroxyphenyl, 3,5-dihydroxyphenyl, 2,3,4-trihydroxyphenyl, 2,3,5-trihydroxyphenyl, 2,3,6-trihydroxy-phenyl, 2,4,6-trihydroxyphenyl, 2,3,4,5-tetrahydroxyphenyl, 2,3,4,6-tetrahydroxyphenyl, 2,3,4,5,6-pentahydroxyphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2,3-dimethoxyphenyl, 2,4-dimethoxyphenyl, 2,5-dimethoxyphenyl, 2,6-dimethoxyphenyl, 3,4-dimethoxy-phenyl, 3,5-dimethoxyphenyl, 2,3,4-trimethoxyphenyl, 2,3,5-trimethoxyphenyl, 2,3,6-trimethoxyphenyl, 2,4,6-trimethoxyphenyl, 2,3,4,5-tetramethoxyphenyl, 2,3,4,6-tetra-methoxyphenyl, 2,3,4,5,6-pentamethoxyphenyl, 2-aminophenyl, 3-aminophenyl, 4-aminophenyl, 2,3-diaminophenyl, 2,4-di-aminophenyl, 2,5-diaminophenyl, 2,6-diaminophenyl, 3,4-diaminophenyl, 3,5-diamino-phenyl, 2,3,4-triaminophenyl, 2,3,5-triaminophenyl, 2,3,6-triaminophenyl, 2,4,6-tri-aminophenyl, 2,3,4,5-tetraaminophenyl, 2,3,4,6-tetraaminophenyl, 2,3,4,5,6-penta-aminophenyl, 2-(dimethylamino)phenyl, 3-(dimethylamino)phenyl, 4-(dimethylamino)phenyl, 2,3-di(dimethylamino)phenyl, 2,4-di(dimethylamino)-phenyl, 2,5-di(dimethylamino)-phenyl, 2,6-di(dimethylamino)phenyl, 3,4-di(dimethylamino)phenyl, 3,5-di(dimethyl-amino)phenyl, 2,3,4-tri(dimethyl-amino)phenyl, 2,3,5-tri(dimethylamino)phenyl, 2,3,6-tri(dimethylamino)phenyl, 2,4,6-tri(dimethylamino)phenyl, 2,3,4,5-tetra(dimethylamino)-phenyl, 2,3,4,6-tetra(dimethylamino)phenyl, and 2,3,4,5,6-penta(dimethylamino)phenyl, and a pharmaceutically acceptable salt thereof.
 2. The method of claim 1, wherein the inflammatory condition is selected from the group consisting of asthma, rheumatoid arthritis, reactive arthritis, spondylarthritis, systemic vasculitis, insulin dependent diabetes mellitus, multiple sclerosis, experimental allergic encephalomyelitis, Sjögren's syndrome, graft versus host disease, inflammatory bowel disease including Crohn's disease, ulcerative colitis, ischemia reperfusion injury, Alzheimer's disease, transplant rejection (allogeneic and xenogeneic), thermal trauma, any immune complex-induced inflammation, glomerulonephritis, myasthenia gravis, cerebral lupus, Guillaine-Barre syndrome, vasculitis, systemic sclerosis, anaphylaxis, catheter reactions, atheroma, infertility, thyroiditis, ARDS, post-bypass syndrome, hemodialysis, juvenile rheumatoid, Behcets syndrome, hemolytic anemia, pemphigus, bulbous pemphigoid, stroke, atherosclerosis, and scleroderma.
 3. The method of claim 1, wherein the disease or condition associated with heart malfunction is selected from the group consisting of myocardial infarction, restenosis, hypertension, primary cardiomyopathy and secondary cardiomyopathy, wherein the primary and secondary cardiomyopathy is selected from the group consisting of dilated cardiomyopathy hypertrophic cardiomyopathy, and restrictive cardiomyopathy, further wherein the hypertrophic cardiomyopathy is selected from the group consisting of ischemic, non-ischemic, idiopathic, congestive, diabetic, peripartium, alcoholic, viral, and valvular.
 4. The method of claim 1, wherein the disease or condition affecting the vasculature is selected from the group consisting of peripheral vascular disease, atherosclerosis, restenosis, and hypertension. 